Modified Factor VII

ABSTRACT

The catalytic active site of Factor VII is modified to produce a compound which effectively interrupts the blood coagulation cascade. The modifications render Factor VIIa substantially unable to activate plasma Factors X or IX. Pharmaceutical compositions of the modified Factor VII are used to treat a variety of coagulation-related disorders.

This application is a divisional of Ser. No. 08/327,690, filed Oct. 24,1994, now U.S. Pat. No. 5,817,788, which is a continuation-in-part ofPCT/US94/05779, filed May 23, 1994, and a continuation-in-part of Ser.No. 08/065,725, filed May 21, 1993, now abandoned, which is acontinuation-in-part of PCT/US92/01636, filed Feb. 28, 1992 and adivisional of Ser. No. 07/662,920, filed Feb. 28, 1991, abandoned andnow Ser. No. 08/164,666, now abandoned, each of which is expresslyincorporated herein in its entirety by reference.

FIELD OF THE INVENTION

The present invention relates to proteins useful as anticoagulants. Morespecifically, the present invention relates to modified forms of FactorVII that inhibit blood coagulation and tissue factor.

BACKGROUND OF THE INVENTION

Blood coagulation is a process consisting of a complex interaction ofvarious blood components, or factors, which eventually gives rise to afibrin clot. Generally, the blood components which participate in whathas been referred to as the coagulation "cascade" are proenzymes orzymogens, enzymatically inactive proteins which are converted toproteolytic enzymes by the action of an activator, itself an activatedclotting factor. Coagulation factors that have undergone such aconversion and generally referred to as "active factors," and aredesignated by the addition of a lower case "a" suffix (e.g., FactorVIIa).

Activated Factor X ("Xa") is required to convert prothrombin tothrombin, which then converts fibrinogen to fibrin as a final stage informing a fibrin clot. There are two systems, or pathways, that promotethe activation of Factor X. The "intrinsic pathway" refers to thosereactions that lead to thrombin formation through utilization of factorspresent only in plasma. A series of protease-mediated activationsultimately generates Factor IXa which, in conjunction with Factor VIIIa,cleaves Factor X into Xa. An identical proteolysis is effected by FactorVIIa and its co-factor, tissue factor, in the "extrinsic pathway" ofblood coagulation. Tissue factor is a membrane bound protein and doesnot normally circulate in plasma. Upon vessel disruption, however, itcan complex with Factor VIIa to catalyze Factor X activation or FactorIX activation in the presence of Ca++and phospholipid (Nemerson andGentry, Biochem. 25:4020-4033 (1986)). While the relative importance ofthe two coagulation pathways in hemostasis is unclear, in recent yearsFactor VII and tissue factor have been found to play a pivotal role inthe regulation of blood coagulation.

Factor VII is a trace plasma glycoprotein that circulates in blood as asingle-chain zymogen. The zymogen is catalytically inactive (Williams etal., J. Biol. Chew. 264:7536-7543 (1989); Rao et al., Proc. Natl. Acad.Sci. USA. 85:6687-6691 (1988)). Single-chain Factor VII may be convertedto two-chain Factor VIIa by Factor Xa, Factor XIIa, Factor IXa orthrombin in vitro. Factor Xa is believed to be the major physiologicalactivator of Factor VII. Like several other plasma proteins involved inhemostasis, Factor VII is dependent on vitamin K for its activity, whichis required for the γ-carboxylation of multiple glutamic acid residuesthat are clustered in the amino terminus of the protein. Theseγ-carboxylated glutamic acids are required for the metal-associatedinteraction of Factor VII with phospholipids.

The conversion of zymogen Factor VII into the activated two-chainmolecule occurs by cleavage of an internal peptide bond locatedapproximately in the middle of the molecule. In human Factor VII, theactivation cleavage site is at Arg₁₅₂ -Ile₁₅₃ (Hagen et al., Proc. Natl.Acad. Sci. USA 83: 2412-2416 (1986); Thim et al., Biochem. 27:7785-7793(1988) both of which are incorporated herein by references). Bovinefactor VII is activated by cleavage at the analogous Arg₁₅₂ -Ile₁₅₃ bond(Takeya et al., J. Biol. Chem. 263: 14868-14877, 1988). In the presenceof tissue factor, phospholipids and calcium ions, the two-chain FactorVIIa rapidly activates Factor X or Factor IX by limited proteolysis.

It is often necessary to selectively block the coagulation cascade in apatient. Anticoagulants such as heparin, coumarin, derivatives ofcoumarin, indandione derivatives, or other agents may be used, forexample, during kidney dialysis, or to treat deep vein thrombosis,disseminated intravascular coagulation (DIC), and a host of othermedical disorders. For example, heparin treatment or extracorporealtreatment with citrate ion (U.S. Pat. No. 4,500,309) may be used indialysis to prevent coagulation during the course of treatment. Heparinis also used in preventing deep vein thrombosis in patients undergoingsurgery.

Treatment with heparin and other anticoagulants may, however, haveundesirable side effects. Available anticoagulants generally actthroughout the body, rather than acting specifically at a clot site.Heparin, for example, may cause heavy bleeding. Furthermore, with ahalf-life of approximately 80 minutes, heparin is rapidly cleared fromthe blood, necessitating frequent administration. Because heparin actsas a cofactor for antithrombin III (AT III), and AT III is rapidlydepleted in DIC treatment, it is often difficult to maintain the properheparin dosage, necessitating continuous monitoring of AT III andheparin levels. Heparin is also ineffective if AT III depletion isextreme. Further, prolonged use of heparin may also increase plateletaggregation and reduce platelet count, and has been implicated in thedevelopment of osteoporosis. Indandione derivatives may also have toxicside effects.

In addition to the anticoagulants briefly described above, severalnaturally occurring proteins have been found to have anticoagulantactivity. For example, Reutelingsperger (U.S. Pat. No. 4,736,018)isolated anticoagulant proteins from bovine aorta and human umbilicalvein arteries. Maki et al. (U.S. Pat. No. 4,732,891) disclose humanplacenta-derived anticoagulant proteins. In addition, AT III has beenproposed as a therapeutic anticoagulant (Schipper et al., Lancet 1(8069): 854-856 (1978); Jordan, U.S. Pat. No. 4,386,025; Bock et al.,U.S. Pat. No. 4,517,294).

Proliferation of smooth muscle cells (SMCs) in the vessel wall is animportant event in the formation of vascular lesions in atherosclerosis,after vascular reconstruction or in response to other vascular injury.For example, treatment of atherosclerosis frequently includes theclearing of blocked vessels by angioplasty, endarterectomy or reductionatherectomy, or by bypass grafting, surgical procedures in whichatherosclerotic plaques are compressed or removed throughcatheterization (angioplasty), stripped away from the arterial wallthrough an incision (endarterectomy) or bypassed with natural orsynthetic grafts. These procedures remove the vascular endothelium,disturb the underlying intimal layer, and result in the death of medialSMCs. This injury is followed by medial SMC proliferation and migrationinto the intima, which characteristically occurs within the first fewweeks and up to six months after injury and stops when the overlyingendothelial layer is reestablished. In humans, these lesions arecomposed of about 20% cells and 80% extracellular matrix.

In about 30% or more of patients treated by angioplasty, endarterectomyor bypass grafts, thrombosis and/or SMC proliferation in the intimacauses re-occlusion of the vessel and consequent failure of thereconstructive surgery. This closure of the vessel subsequent to surgeryis known as restenosis.

There is still a need in the art for improved compositions havinganticoagulant activity which can be administered at relatively low dosesand do not produce the undesirable side effects associated withtraditional anticoagulant compositions. The present invention fulfillsthis need by providing anticoagulants that act specifically at sites ofinjury, and further provides other related advantages.

SUMMARY OF THE INVENTION

Novel compositions which comprise modified Factor VII havinganticoagulant properties are provided. The modified Factor VIIcompositions also inhibit tissue factor. The Factor VII sequence has atleast one amino acid modification, where the modification is selected soas to substantially reduce the ability of activated Factor VII tocatalyze the activation of plasma Factors X or IX, and thus is capableof inhibiting clotting activity. The novel Factor VII has an active sitemodified by at least one amino acid substitution, and in its modifiedform is capable of binding tissue factor. The modified Factor VIIcompositions are typically in substantially pure form.

The compositions of the invention are particularly useful in methods fortreating patients when formulated into pharmaceutical compositions,where they may be given to individuals suffering from a variety ofdisease states to treat coagulation-related conditions. Such modifiedFactor VII molecules, capable of binding tissue factor but having asubstantially reduced ability to catalyze activation of other factors inthe clotting cascade, may possess a longer plasma half-life and thus acorrespondingly longer period of anticoagulative activity when comparedto other anticoagulants. Among the medical indications for the subjectcompositions are those commonly treated with anticoagulants, such as,for example, deep vein thrombosis, pulmonary embolism, stroke,disseminated intravascular coagulation (DIC), fibrin deposition in lungsand kidneys associated with gram-negative endotoxemia, and myocardialinfarction. The compositions can be used to inhibit vascular restenosisas occurs following mechanical vascular injury, such as injury caused byballoon angioplasty, endarterectomy, reductive atherectomy, stentplacement, laser therapy or rotablation, or as occurs secondary tovascular grafts, stents, bypass grafts or organ transplants. Thecompositions can thus be used to inhibit platelet deposition andassociated disorders. Thus, a method of inhibiting coagulaticn, vascularrestenosis or platelet deposition, for example, comprises administeringto a patient a composition comprising modified Factor VII, such as thathaving at least one amino acid substitution in a catalytic triad ofSer₃₄₄, Asp₂₄₂ and His₁₉₃, in an amount sufficient to effectivelyinhibit coagulation, vascular restenosis or platelet deposition. Themethods also find use in the treatment of acute closure of a coronaryartery in an individual, which comprises administering the modifiedFactor VII in conjunction with tissue plasminogen activator orstreptokinase.

Typically, for administration to humans the pharmaceutical compositionswill comprise modified human Factor VII protein andpharmaceutically-acceptable carriers and buffers.

In preferred embodiments of human and bovine Factor VII, the active siteresidue Ser₃₄₄ is modified, replaced with Gly, Met, Thr, or morepreferably, Ala. Such substitution could be made separately or incombination with substitution(s) at other sites in the catalytic triad,which includes His₁₉₃ and Asp₂₄₂.

In another aspect the invention relates to a polynucleotide moleculecomprising two operatively linked sequence coding regions encoding,respectively, a pre-pro peptide and a gla domain of a vitaminK-dependent plasma protein, and a gla domain-less Factor VII protein,wherein upon expression said polynucleotide encodes a modified FactorVII molecule which does not significantly activate plasma Factors X orIX, and is capable of binding tissue factor. The modified Factor VIImolecule expressed by this polynucleotide is a biologically activeanticoagulant, that is, it is capable of inhibiting the coagulationcascade and thus the formation of a fibrin deposit or clot. To expressthe modified Factor VII the polynucleotide molecule is transfected intomammalian cell lines, such as, for example, BHK, BHK 570 or 293 celllines.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 illustrates the construction of an expression vector for a Ser₃₄₄→Ala modified Factor VII DNA sequence. Symbols used include 0-1, the 0-1map unit sequence from adenovirus 5; E, the SV40 enhancer; MLP, theadenovirus 2 major late promotor; SS, a set of splice sites; and pA, thepolyadenylation signal from SV40 in the late orientation.

FIG. 2 shows the effect of bolus injection of DEGR-Factor VIIa onthrombus formation (platelet deposition) on endarterectomized baboonaorta when compared to saline-treated controls. The arteries weremeasured over 60 minutes. The DEGR-Factor VIIa significantly inhibitedthe development of platelet-rich thrombi in this primate model of acutevascular injury.

FIG. 3 shows results obtained when baboon smooth muscle cells wereincubated with increasing concentrations of either FVIIa (open box), orDEGR-FVIIa in the presence of a constant amount of FVIIa (5 nM) (closedbox). The level of FX activation was subsequently determined using thechromogenic substrate S-2222. The data are presented as the amidolyticactivity as a percentage of the activity generated in the presence of 5nM FVIIa alone.

FIG. 4 depicts the size of the intimal area of baboons following carotidartery endarterectomy and treatment with DEGR-Factor VIIa for 7 or 30days, compared to control animals.

FIG. 5 illustrates the ratio of the intimal area to the intimal+mediaarea of baboon femoral artery following balloon injury and treatmentwith DEGR-Factor VIIa, where the control group included 5 vessels, 7 daytreatment examined 11 vessels, and 30 day treatment examined 2 vessels(n=number of vessels examined).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Novel modified Factor VII having anticoagulant activity is provided bythe present invention. The modified Factor VII can be in the form of thezymogen (i.e., a single-chain molecule) or can be cleaved at itsactivation site. Thus, by "modified Factor VII" is meant to includemodified Factor VII and modified Factor VIIa molecules that bind tissuefactor and inhibit the activation of Factor IX to IXa and Factor X toXa. Compositions of the modified Factor VII are suitable foradministration to a variety of mammals, particularly humans, to inhibitthe coagulation cascade. Modified Factor VII may be administered to apatient in conjunction with or in place of other anticoagulantcompounds.

Factor VII plays an important role in the coagulation cascade,particularly that involving the extrinsic pathway. Present in thecirculating plasma as an inactive single chain zymogen protein, onceactivated, Factor VIa, in combination with tissue factor and calciumions, activates Factor X to Xa and activates Factor IX to IXa, with theeventual formation of a fibrin clot.

The present invention provides the ability to inhibit this sequence ofevents in the coagulation cascade by preventing or otherwise inhibitingthe activation of Factors X and IX by Factor VIIa. Factor VII proteinsof the invention have a catalytic site which is modified to decrease thecatalytic activity of Factor VIIa, while the molecule retains theability to bind to tissue factor. The modified Factor VII moleculescompete with native Factor VII and/or VIIa for binding to tissue factor.As a result, the activation of Factors X and IX is inhibited.

In one aspect of the present invention the catalytic activity of FactorVIIa is inhibited by chemical derivatization of the catalytic center, ortriad. Derivatization may be accomplished by reacting Factor VII with anirreversible inhibitor such as an organophosphor compound, a sulfonylfluoride, a peptide halomethyl ketone or an azapeptide, or by acylation,for example. Preferred peptide halomethyl ketones include PPACK(D-Phe-Pro-Arg chloromethyl-ketone; see U.S. Pat. No. 4,318,904,incorporated herein by reference), D-Phe-Phe-Arg chloromethylketone; andDEGRck (dansyl-GlU-Gly-Arg chloromethylketone).

In another aspect, the catalytic activity of Factor VIIa can also beinhibited by substituting, inserting or deleting amino acids. Inpreferred embodiments amino acid substitutions are made in the aminoacid sequence of the Factor VII catalytic triad, defined herein as theregions which contain the amino acids which contribute to the FactorVIIa catalytic site. The substitutions, insertions or deletions in thecatalytic triad are generally at or adjacent to the amino acids whichform the catalytic site. In the human and bovine Factor VII proteins,the amino acids which form a catalytic "triad" are Ser₃₄₄, Asp₂₄₂, andHis₁₉₃ (subscript numbering indicating position in the sequence). Thecatalytic sites in Factor VII from other mammalian species may bedetermined using presently available techniques including, among others,protein isolation and amino acid sequence analysis. Catalytic sites mayalso be determined by aligning a sequence with the sequence of otherserine proteases, particularly chymotrypsin, whose active site has beenpreviously determined (Sigler et al., J. Mol. Biol., 35:143-164 (1968),incorporated herein by reference), and therefrom determining from saidalignment the analogous active site residues.

The amino acid substitutions, insertions or deletions are made so as toprevent or otherwise inhibit activation by the Factor VIa of Factors Xand/or IX. The Factor VII so modified should, however, also retain theability to compete with authentic Factor VII and/or Factor VIIa forbinding to tissue factor in the coagulation cascade. Such competitionmay readily be determined by means of, e.g., a clotting assay asdescribed herein, or a competition binding assay using, e.g., a cellline having cell-surface tissue factor, such as the human bladdercarcinoma cell line J82 (Sakai et al. J. Biol. Chem. 264: 9980-9988(1989), incorporated by reference herein.) The amino acids which formthe catalytic site in Factor VII, such as Ser₃₄₄, Asp₂₄₂, and His₁₉₃ inhuman and bovine Factor VII, may either be substituted or deleted.Within the present invention, it is preferred to change only a singleamino acid, thus minimizing the likelihood of increasing theantigenicity of the molecule or inhibiting its ability to bind tissuefactor, however two or more amino acid changes (substitutions, additionsor deletions) may be made and combinations of substitution(s),addition(s) and deletions) may also be made. In a preferred embodimentfor human and bovine Factor VII, Ser₃₄₄ is preferably substituted withAla, but Gly, Met, Thr or other amino acids can be substituted. It ispreferred to replace Asp with Glu and to replace His with Lys or Arg. Ingeneral, substitutions are chosen to disrupt the tertiary proteinstructure as little as possible. The model of Dayhoff et al. (in Atlasof Protein Structure 1978, Nat'l Biomed. Res. Found., Washington, D.C.),incorporated herein by reference, may be used as a guide in selectingother amino acid substitutions. One may introduce residue alterations asdescribed above in the catalytic site of appropriate Factor VII sequenceof human, bovine or other species and test the resulting protein for adesired level of inhibition of catalytic activity and resultinganticoagulant activity as described herein. For the modified Factor VIIthe catalytic activity will be substantially inhibited, generally lessthan about 5% of the catalytic activity of wild-type Factor VII of thecorresponding species, more preferably less than about 1%.

The proteins of the present invention may be produced through the use ofrecombinant DNA techniques. In general, a cloned wild-type Factor VIIDNA sequence is modified to encode the desired protein. This modifiedsequence is then inserted into an expression vector, which is in turntransformed or transfected into host cells. Higher eukaryotic cells, inparticular cultured mammalian cells, are preferred as host cells. Thecomplete nucleotide and amino acid sequences for human Factor VII areknown. See U.S. Pat. No. 4,784,950, which is incorporated herein byreference, where the cloning and expression of recombinant human FactorVII is described. The bovine Factor VII sequence is described in Takeyaet al., J. Biol. Chem, 263:14868-14872 (1988), which is incorporated byreference herein.

The amino acid sequence alterations may be accomplished by a variety oftechniques. Modification of the DNA sequence may be by site-specificmutagenesis. Techniques for site-specific mutagenesis are well known inthe art and are described by, for example, Zoller and Smith (DNA3:479-488, 1984). Thus, using the nucleotide and amino acid sequences ofFactor VII, one may introduce the alterations) of choice.

The Factor VII modified according to the present invention includesthose proteins that have the amino-terminal portion (gla domain)substituted with a gla domain of one of the vitamin K-dependent plasmaproteins Factor IX, Factor X, prothrombin, protein C, protein S orprotein Z. The gla domains of the vitamin K-dependent plasma proteinsare characterized by the presence of gamma-carboxy glutamic acidresidues and are generally from about 30 to about 40 amino acids inlength with C-termini corresponding to the positions of exon-intronboundaries in the respective genes. Methods for producing Factor VIIwith a heterologous gla domain are disclosed in U.S. Pat. No. 4,784,950,incorporated by reference herein.

DNA sequences for use within the present invention will typically encodea pre-pro peptide at the amino-terminus of the Factor VII protein toobtain proper post-translational processing (e.g. gamma-carboxylation ofglutamic acid residues) and secretion from the host cell. The pre-propeptide may be that of Factor VII or another vitamin K-dependent plasmaprotein, such as Factor IX, Factor X, prothrombin, protein C or proteinS. As will be appreciated by those skilled in the art, additionalmodifications can be made in the amino acid sequence of the modifiedFactor VII where those modifications do not significantly impair theability of the protein to act as an anticoagulant. For example, theFactor VII modified in the catalytic triad can also be modified in theactivation cleavage site to inhibit the conversion of zymogen Factor VIIinto its activated two-chain form, as generally described in U.S. Pat.No. 5,288,629, incorporated herein by reference.

Expression vectors for use in carrying out the present invention willcomprise a promoter capable of directing the transcription of a clonedgene or cDNA. Preferred promoters for use in cultured mammalian cellsinclude viral promoters and cellular promoters. Viral promoters includethe SV40 promoter (Subramani et al., Mol. Cell. Diol. 1:854-864, 1981)and the CMV promoter (Boshart et al., Cell 41:521-530, 1985). Aparticularly preferred viral promoter is the major late promoter fromadenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol, 2:1304-1319, 1982).Cellular promoters include the mouse kappa gene promoter (Bergman etal., Proc. Natl. Acad. Sci. USA 81:7041-7045, 1983) and the mouse V_(H)promoter (Loh et al., Cell 33:85-93, 1983). A particularly preferredcellular promoter is the mouse metallothionein-I promoter (Palmiter etal., Science 222:809-814, 1983). Expression vectors may also contain aset of RNA splice sites located downstream from the promoter andupstream from the insertion site for the Factor VII sequence itself.Preferred RNA splice sites may be obtained from adenovirus and/orimmunoglobulin genes. Also contained in the expression vectors is apolyadenylation signal located downstream of the insertion site.Particularly preferred polyadenylation signals include the early or latepolyadenylation signal from SV40 (Kaufman and Sharp, ibid.), thepolyadenylation signal from the adenovirus 5 Elb region, the humangrowth hormone gene terminator (DeNoto et al. Nuc. Acids Res.9:3719-3730, 1981) or the polyadenylation signal from the human FactorVII gene or the bovine Factor VII gene. The expression vectors may alsoinclude a noncoding viral leader sequence, such as the adenovirus 2tripartite leader, located between the promoter and the RNA splicesites; and enhancer sequences, such as the SV40 enhancer.

Cloned DNA sequences are introduced into cultured mammalian cells by,for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics7:603-616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973) orelectroporation (Neumann et al., EMBO J. 1:841-845, 1982). To identifyand select cells that express the exogenous DNA, a gene that confers aselectable phenotype (a selectable marker) is generally introduced intocells along with the gene or cDNA of interest. Preferred selectablemarkers include genes that confer resistance to drugs such as neomycin,hygromycin, and methotrexate. The selectable marker may be anamplifiable selectable marker. A preferred amplifiable selectable markeris a dihydrofolate reductase (DHFR) sequence. Selectable markers arereviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers,Stoneham, Mass., incorporated herein by reference). The choice ofselectable markers is well within the level of ordinary skill in theart.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid. If on the same plasmid, the selectable marker and thegene of interest may be under the control of different promoters or thesame promoter, the latter arrangement producing a dicistronic message.Constructs of this type are known in the art (for example, Levinson andSimonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to addadditional DNA, known as "carrier DNA," to the mixture that isintroduced into the cells.

After the cells have taken up the DNA, they are grown in an appropriategrowth medium, typically 1-2 days, to begin expressing the gene ofinterest. As used herein the term "appropriate growth medium" means amedium containing nutrients and other components required for the growthof cells and the expression of the modified Factor VII gene. Mediagenerally include a carbon source, a nitrogen source, essential aminoacids, essential sugars, vitamins, salts, phospholipids, protein andgrowth factors. For production of gamma-carboxylated modified FactorVII, the medium will contain vitamin K, preferably at a concentration ofabout 0.1 μg/ml to about 5 μg/ml. Drug selection is then applied toselect for the growth of cells that are expressing the selectable markerin a stable fashion. For cells that have been transfected with anamplifiable selectable marker the drug concentration may be increased toselect for an increased copy number of the cloned sequences, therebyincreasing expression levels. Clones of stably transfected cells arethen screened for expression of modified Factor VII.

Preferred mammalian cell lines for use in the present invention includethe COS-1 (ATCC CRL 1650), baby hamster kidney (BHK) and 293 (ATCC CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines. Apreferred BHK cell line is the tk⁻ ts13 BHK cell line (Waechter andBaserga, Proc. Natl. Acad. Sci. USA 79:1106-1110, 1982, incorporatedherein by reference), hereinafter referred to as BHK 570 cells. The BHK570 cell line has been deposited with the American Type CultureCollection, 12301 Parklawn Dr., Rockville, Md. 20852, under ATCCaccession number CRL 10314. A tk⁻ ts13 BHK cell line is also availablefrom the ATCC under accession number CRL 1632. In addition, a number ofother cell lines may be used within the present invention, including RatHep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCCCCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and Chasin, Proc.Natl. Acad. Sci. USA 77:4216-4220, 1980).

Within the present invention, transgenic animal technology may beemployed to produce modified Factor VII. It is preferred to produce theproteins within the mammary glands of a host female mammal. Expressionin the mammary gland and subsequent secretion of the protein of interestinto the milk overcomes many difficulties encountered in isolatingproteins from other sources. Milk is readily collected, available inlarge quantities, and well characterized biochemically. Furthermore, themajor milk proteins are present in milk at high concentrations(typically from about 1 to 15 g/l). From a commercial point of view, itis clearly preferable to use as the host a species that has a large milkyield. While smaller animals such as mice and rats can be used (and arepreferred at the proof of principle stage), within the present inventionit is preferred to use livestock mammals including, but not limited to,pigs, goats, sheep and cattle. Sheep are particularly preferred due tosuch factors as the previous history of transgenesis in this species,milk yield, cost and the ready availability of equipment for collectingsheep milk. See WIPO Publication WO 88/00239 for a comparison of factorsinfluencing the choice of host species. It is generally desirable toselect a breed of host animal that has been bred for dairy use, such asEast Friesland sheep, or to introduce dairy stock by breeding of thetransgenic line at a later date. In any event, animals of known, goodhealth status should be used.

To obtain expression in the mammary gland, a transcription promoter froma milk protein gene is used. Milk protein genes include those genesencoding caseins (see U.S. Pat. No. 5,304,489, incorporated herein byreference), beta-lactoglobulin, a-lactalbumin, and whey acidic protein.The beta-lactoglobulin (BLG) promoter is preferred. In the case of theovine beta-lactoglobulin gene, a region of at least the proximal 406 bpof 5' flanking sequence of the gene will generally be used, althoughlarger portions of the 5' flanking sequence, up to about 5 kbp, arepreferred, such as a ˜4.25 kbp DNA segment encompassing the 5' flankingpromoter and non-coding portion of the beta-lactoglobulin gene. SeeWhitelaw et al., Biochem J. 286: 31-39 (1992). Similar fragments ofpromoter DNA from other species are also suitable.

Other regions of the beta-lactoglobulin gene may also be incorporated inconstructs, as may genomic regions of the gene to be expressed. It isgenerally accepted in the art that constructs lacking introns, forexample, express poorly in comparison with those that contain such DNAsequences (see Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836-840(1988); Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478-482 (1991);Whitelaw et al., Transgenic Res. 1: 3-13 (1991); WO 89/01343; and WO91/02318, each of which is incorporated herein by reference). In thisregard, it is generally preferred, where possible, to use genomicsequences containing all or some of the native introns of a geneencoding the protein or polypeptide of interest, thus the furtherinclusion of at least some introns from, e.g, the beta-lactoglobulingene, is preferred. One such region is a DNA segment which provides forintron splicing and RNA polyadenylation from the 3' non-coding region ofthe ovine beta-lactoglobulin gene. When substituted for the natural 3'non-coding sequences of a gene, this ovine beta-lactoglobulin segmentcan both enhance and stabilize expression levels of the protein orpolypeptide of interest. Within other embodiments, the regionsurrounding the initiation ATG of the modified Factor VII sequence isreplaced with corresponding sequences from a milk specific protein gene.Such replacement provides a putative tissue-specific initiationenvironment to enhance expression. It is convenient to replace theentire modified Factor VII pre-pro and 5' non-coding sequences withthose of, for example, the BLG gene, although smaller regions may bereplaced.

For expression of modified Factor VII in transgenic animals, a DNAsegment encoding modified Factor VII is operably linked to additionalDNA segments required for its expression to produce expression units.Such additional segments include the above-mentioned promoter, as wellas sequences which provide for termination of transcription andpolyadenylation of mRNA. The expression units will further include a DNAsegment encoding a secretory signal sequence operably linked to thesegment encoding modified Factor VII. The secretory signal sequence maybe a native Factor VII secretory signal sequence or may be that ofanother protein, such as a milk protein. See, for example, von Heinje,Nuc. Acids Res. 14: 4683-4690 (1986); and Meade et al., U.S. Pat. No.4,873,316, which are incorporated herein by reference.

Construction of expression units for use in transgenic animals isconveniently carried out by inserting a modified Factor VII sequenceinto a plasmid or phage vector containing the additional DNA segments,although the expression unit may be constructed by essentially anysequence of ligations. It is particularly convenient to provide a vectorcontaining a DNA segment encoding a milk protein and to replace thecoding sequence for the milk protein with that of a modified Factor VIIpolypeptide, thereby creating a gene fusion that includes the expressioncontrol sequences of the milk protein gene. In any event, cloning of theexpression units in plasmids or other vectors facilitates theamplification of the modified Factor VII sequence. Amplification isconveniently carried out in bacterial (e.g. E. coli) host cells, thusthe vectors will typically include an origin of replication and aselectable marker functional in bacterial host cells.

The expression unit is then introduced into fertilized eggs (includingearly-stage embryos) of the chosen host species. Introduction ofheterologous DNA can be accomplished by one of several routes, includingmicroinjection (e.g. U.S. Pat. No. 4,873,191), retroviral infection(Jaenisch, Science 240: 1468-1474 (1988)) or site-directed integrationusing embryonic stem (ES) cells (reviewed by Bradley et al.,Bio/Technology 10: 534-539 (1992)). The eggs are then implanted into theoviducts or uteri of pseudopregnant females and allowed to develop toterm. Offspring carrying the introduced DNA in their germ line can passthe DNA on to their progeny in the normal, Mendelian fashion, allowingthe development of transgenic herds.

General procedures for producing transgenic animals are known in theart. See, for example, Hogan et al., Manipulating the Mouse Embryo: ALaboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al.,Bio/Technology 6: 179-183 (1988); Wall et al., Biol. Reprod. 32: 645-651(1985); Buhler et al., Bio/Technology 8: 140-143 (1990); Ebert et al.,Bio/Technology 9: 835-838 (1991); Krimpenfort et al., Bio/Technology 9:844-847 (1991); Wall et al., J. Cell. Biochem. 49: 113-120 (1992); U.S.Pat. Nos. 4,873,191 and 4,873,316; WIPO publications WO 88/00239, WO90/05188, WO 92/11757; and GB 87/00458, which are incorporated herein byreference. Techniques for introducing foreign DNA sequences into mammalsand their germ cells were originally developed in the mouse. See, e.g.,Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380-7384 (1980); Gordonand Ruddle, Science 214: 1244-1246 (1981); Palmiter and Brinster, Cell41: 343-345 (1985); Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 (1985); and Hogan et al. (ibid.). These techniques weresubsequently adapted for use with larger animals, including livestockspecies (see e.g., WIPO publications WO 88/00239, WO 90/05188, and WO92/11757; and Simons et al., Bio/Technology 6: 179-183 (1988). Tosummarize, in the most efficient route used to date in the generation oftransgenic mice or livestock, several hundred linear molecules of theDNA of interest are injected into one of the pro-nuclei of a fertilizedegg according to established techniques. Injection of DNA into thecytoplasm of a zygote can also be employed. Production in transgenicplants may also be employed. Expression may be generalized or directedto a particular organ, such as a tuber. See, Hiatt, Nature 344:469-479(1990); Edelbaum et al., J. Interferon Res. 12:449-453 (1992); Sijmonset al., Bio/Technology 8:217-221 (1990); and European Patent OfficePublication EP 255,378.

Modified Factor VII produced according to the present invention may bepurified by affinity chromatography on an anti-Factor VII antibodycolumn. The use of calcium-dependent monoclonal antibodies, as describedby Wakabayashi et al., J. Biol. Chem, 261:11097-11108, (1986) and Thimet al., Biochem. 27: 7785-7793, (1988), incorporated by referenceherein, is particularly preferred. Additional purification may beachieved by conventional chemical purification means, such as highperformance liquid chromatography. Other methods of purification,including barium citrate precipitation, are known in the art, and may beapplied to the purification of the novel modified Factor VII describedherein (see, generally, Scopes, R., Protein Purification,Springer-Verlag, N.Y., 1982). Substantially pure modified Factor VII ofat least about 90 to 95% homogeneity is preferred, and 98 to 99% or morehomogeneity most preferred, for pharmaceutical uses. Once purified,partially or to homogeneity as desired, the modified Factor VII may thenbe used therapeutically.

Within one embodiment of the invention the modified Factor VII iscleaved at its activation site to convert it to its two-chain form.Activation may be carried out according to procedures known in the art,such as those disclosed by Osterud, et al., Biochemistry 11:2853-2857(1972); Thomas, U.S. Pat. No. 4,456,591; Hedner and Kisiel, J. Clin.Invest. 71:1836-1841 (1983); or Kisiel and Fujikawa, Behring Inst. Mitt.73:29-42 (1983), which are incorporated herein by reference. Theresulting molecule is then formulated and administered as describedbelow.

The modified Factor VII molecules of the present invention andpharmaceutical compositions thereof are particularly useful foradministration to humans to treat a variety of conditions involvingintravascular coagulation. For example, although deep vein thrombosisand pulmonary embolism can be treated with conventional anticoagulants,the modified Factor VII described herein may be used to prevent theoccurrence of thromboembolic complications in identified high riskpatients, such as those undergoing surgery or those with congestiveheart failure. In addition, modified Factor VII may act as an antagonistfor tissue factor-mediated induction of coagulation, thus blocking theproduction of thrombin and the subsequent deposition of fibrin. As such,modified Factor VII may be useful for inhibiting tissue factor activityresulting in, for example, the inhibition of blood coagulation,thrombosis or platelet deposition.

The modified Factor VII molecules of the present invention may beparticularly useful in the treatment of intimal hyperplasia orrestenosis due to acute vascular injury. Acute vascular injuries arethose which occur rapidly (i.e. over days to months), in contrast tochronic vascular injuries (e.g. atherosclerosis) which develop over alifetime. Acute vascular injuries often result from surgical proceduressuch as vascular reconstruction, wherein the techniques of angioplasty,endarterectomy, atherectomy, vascular graft emplacement or the like areemployed. Hyperplasia may also occur as a delayed response in responseto, e.g., graft emplacement or organ transplantation. Since modifiedFactor VII is more selective than heparin, generally binding only tissuefactor which has been exposed at sites of injury, and because modifiedFactor VII does not destroy other coagulation proteins, it will be moreeffective and less likely to cause bleeding complications than heparinwhen used prophylactically for the prevention of deep vein thrombosis.The dose of modified Factor VII for prevention of deep vein thrombosisis in the range of about 50 μg to 500 mg/day, more typically 1 mg to 200mg/day, and more preferably 10 to about 175 mg/day for a 70 kg patient,and administration should begin at least about 6 hours prior to surgeryand continue at least until the patient becomes ambulatory. The dose ofmodified Factor VII in the treatment for restenosis will vary with eachpatient but will generally be in the range of those suggested above.

Recent advances in the treatment of coronary vascular disease includethe use of mechanical interventions to either remove or displaceoffending plaque material in order to re-establish adequate blood flowthrough the coronary arteries. Despite the use of multiple forms ofmechanical interventions, including balloon angioplasty, reductionatherectomy, placement of vascular stents, laser therapy, or rotoblator,the effectiveness of these techniques remains limited by anapproximately 40% restenosis rate within 6 months after treatment.

Restenosis is thought to result from a complex interaction of biologicalprocesses including platelet deposition and thrombus formation, releaseof chemotactic and mitogenic factors, and the migration andproliferation of vascular smooth muscle cells into the intima of thedilated arterial segment.

The inhibition of platelet accumulation at sites of mechanical injurycan limit the rate of restenosis in human subjects. Therapeutic use of amonoclonal antibody to platelet GpIIb/IIIa is able to limit the level ofrestenosis in human subjects (Califf et al., N. Engl. J. Med.,330:956-961 (1994)). The antibody is able to bind to the GpIIb/IIIareceptor on the surfaces of platelets and thereby inhibit plateletaccumulation. This data suggests that inhibition of plateletaccumulation at the site of mechanical injury in human coronary arteriesis beneficial for the ultimate healing response that occurs. Whileplatelet accumulation occurs at sites of acute vascular injuries, thegeneration of thrombin at these sites may be responsible for theactivation of the platelets and their subsequent accumulation.

As shown in the examples that follow, the modified Factor VII of thepresent invention is able to bind to cell-surface tissue factor. Forexample, DEGR-Factor VIIa binds cell-surface tissue factor with anequivalent or higher affinity than wild-type Factor VIIa. DEGR-FactorVIIa, however, has no enzymatic activity, yet it binds to tissue factorand acts as a competitive antagonist for wild-type Factor VIa, therebyinhibiting the subsequent steps in the extrinsic pathway of coagulationleading to the generation of thrombin.

Modified Factor VII molecules of the present invention which maintaintissue factor binding inhibit platelet accumulation at the site ofvascular injury by blocking the production of thrombin and thesubsequent deposition of fibrin.

Due to the ability of DEGR-Factor VII to block thrombin generation andlimit platelet deposition at sites of acute vascular injury, modifiedFactor VII molecules which maintain tissue factor binding activity butlack Factor VIIa enzymatic activity can be used to inhibit vascularrestenosis.

Thus, the compositions and methods of the present invention have a widevariety of uses. For example, they are useful in preventing orinhibiting restenosis following intervention, typically mechanicalintervention, to either remove or displace offending plaque material inthe treatment of coronary or peripheral vascular disease, such as inconjunction with and/or following balloon angioplasty, reductiveatherectomy, placement of vascular stents, laser therapy, rotoblation,and the like. The compounds will is typically be administered withinabout 24 hours prior to performing the intervention, and for as much as7 days or more thereafter. Administration can be by a variety of routesas further described herein. The compounds of the present invention canalso be administered systemically or locally for the placement ofvascular grafts (e.g., by coating synthetic or modified natural arterialvascular grafts), at sites of anastomosis, surgical endarterectomy(typically carotid artery endarterectomy), bypass grafts, and the like.The modified Factor VII and VIIa also finds use in inhibiting intimalhyperplasia and accelerated atherosclerosis associated with organtransplantation.

In the treatment of established deep vein thrombosis and/or pulmonaryembolism, the dose of modified Factor VII ranges from about 50 μg to 500mg/day, more typically 1 mg to 200 mg/day, and more preferably 10 mg toabout 175 mg/day for a 70 kg patient as loading and maintenance doses,depending on the weight of the patient and the severity of thecondition. Because of the lower likelihood of bleeding complicationsfrom modified Factor VII infusions, modified Factor VII can replace orlower the dose of heparin during or after surgery in conjunction withthrombectomies or embolectomies.

The modified Factor VII compositions of the present invention will alsohave substantial utility in the prevention of cardiogenic emboli and inthe treatment of thrombotic strokes. Because of its low potential forcausing bleeding complications and its selectivity, modified Factor VIIcan be given to stroke victims and may prevent the extension of theoccluding arterial thrombus. The amount of modified Factor VIIadministered will vary with each patient depending on the nature andseverity of the stroke, but doses will generally be in the range ofthose suggested below.

Pharmaceutical compositions of modified Factor VII provided herein willbe a useful treatment in acute myocardial infarction because of theability of modified Factor VII to inhibit in vivo coagulation. ModifiedFactor VII can be given as an adjuvant with tissue plasminogen activatoror streptokinase during the acute phases of the myocardial infarction.In acute myocardial infarction, the patient is given a loading dose ofat least about 50 μg to 500 mg/day, more typically 1 mg to 200 mg/day,and more preferably 10 mg to about 175 mg/day for a 70 kg patient asloading and maintenance doses.

The modified Factor VII of the present invention is useful in thetreatment of disseminated intravascular coagulation (DIC) and otherconditions associated with gram-negative bacteremia. Patients with DICcharacteristically have widespread microcirculatory thrombi and oftensevere bleeding problems which result from consumption of essentialclotting factors. Because of its selectivity, modified Factor VII willnot aggravate the bleeding problems associated with DIC, as doconventional anticoagulants, but will retard or inhibit the formation ofadditional microvascular fibrin deposits. Thus, the modified Factor VIIof the invention, including the DEGR-Factor VII and DEGR-VIIa, is usefulin the inhibition of fibrin deposition associated with endotoxemia andendotoxin shock and thus also moderates effects associated withgram-negative bacteremia. The DEGR-Factor VIIa has been shown in rabbitsto demonstrate a dose-response effect for blocking fibrin deposition inkidneys and lungs, and in baboons to prolong survival of treatedanimals. In cases of acute bacteremia, endotoxemia or DIC, the patientis given a loading dose of at least about 50 μg to 500 mg/day, moretypically 1 mg to 200 mg/day, and more preferably 10 mg to about 175mg/day for a 70 kg patient, with maintenance doses thereafter in therange of 50 μg to 500 mg/day, typically 1 mg to 200 mg/day for a 70 kgpatient.

The pharmaceutical compositions are intended for parenteral, topical orlocal administration for prophylactic and/or therapeutic treatment.Preferably, the pharmaceutical compositions are administeredparenterally, i.e., intravenously, subcutaneously, or intramuscularly.Thus, this invention provides compositions for parenteral administrationwhich comprise a solution of the modified Factor VII molecules dissolvedin an acceptable carrier, preferably an aqueous carrier. A variety ofaqueous carriers may be used, e.g., water, buffered water, 0.4% saline,0.3% glycine and the like. The modified Factor VII molecules can also beformulated into liposome preparations for delivery or targeting to sitesof injury. Liposome preparations are generally described in, e.g., U.S.Pat. No. 4,837,028, U.S. Pat. No. 4,501,728, and U.S. Pat. No.4,975,282, incorporated herein by reference. The compositions may besterilized by conventional, well known sterilization techniques. Theresulting aqueous solutions may be packaged for use or filtered underaseptic conditions. and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous solution prior to administration. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents and thelike, for example, sodium acetate, sodium lactate, sodium chloride,potassium chloride, calcium chloride, etc. The concentration of modifiedFactor VII in these formulations can vary widely, i.e., from less thanabout 0.5%, usually at or at least about 1% to as much as 15 or 20% byweight and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.

Thus, a typical pharmaceutical composition for intravenous infusioncould be made up to contain 250 ml of sterile Ringer's solution, and 10mg of modified Factor VII. Actual methods for preparing parenterallyadministrable compounds will be known or apparent to those skilled inthe art and are described in more detail in for example, Remington'sPharmaceutical Science, 16th ed., Mack Publishing Company, Easton, Pa.(1982), which is incorporated herein by reference.

The compositions containing the modified Factor VII molecules can beadministered for prophylactic and/or therapeutic treatments. Intherapeutic applications, compositions are administered to a patientalready suffering from a disease, as described above, in an amountsufficient to cure or at least partially arrest the disease and itscomplications. An amount adequate to accomplish this is defined as"therapeutically effective dose." Amounts effective for this use willdepend on the severity of the disease or injury and the weight andgeneral state of the patient, but generally range from about 0.05 mg upto about 500 mg of modified Factor VII per day for a 70 kg patient, withdosages of from about 1.0 mg to about 200 mg of modified Factor VII perday being more commonly used. It must be kept in mind that the materialsof the present invention may generally be employed in serious disease orinjury states, that is, life-threatening or potentially life threateningsituations. In such cases, in view of the minimization of extraneoussubstances and general lack of immunogenicity of modified human FactorVII in humans, it is possible and may be felt desirable by the treatingphysician to administer substantial excesses of these modified FactorVII compositions.

In prophylactic applications, compositions containing the modifiedFactor VII are administered to a patient susceptible to or otherwise atrisk of a disease state or injury to enhance the patient's ownanticoagulative capabilities. Such an amount is defined to be a"prophylactically effective dose." In this use, the precise amountsagain depend on the patient's state of health and weight, but generallyrange from about 0.05 mg to about 500 mg per 70 kilogram patient, morecommonly from about 1.0 mg to about 200 mg per 70 kg of body weight.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. For ambulatory patients requiring daily maintenance levels,the modified Factor VII may be administered by continuous infusion usinga portable pump system, for example.

Local delivery of the modified Factor VII may be carried out, forexample, by way of perfusion, double balloon catheters, stent,incorporated into vascular grafts or stents, hydrogels used to coatballoon catheters, or other well established methods. In any event, thepharmaceutical formulations should provide a quantity of modified FactorVII of this invention sufficient to effectively treat the patient.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLE I Expression of Ser₃₄₄ →Ala₃₄₄ Factor VII

To generate the Ser₃₄₄ →Ala Factor VII active site mutant, plasmidFVII(565+2463)/pDX (U.S. Pat. No. 4,784,950 incorporated herein byreference; deposited with the American Type Culture Collection underaccession number 40205) was digested with Xba I and Kpn I, and theresulting 0.6 kb fragment, comprising the coding region for serine 344,was recovered. This fragment was cloned into Xba I, Kpn I-digestedM13mp19 as shown in the Figure. This manipulation and subsequent stepsdescribed below were generally performed according to standard protocols(as described, for example, by Maniatis et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1982) incorporated herein by reference).

Mutagenesis was carried out on the M13 template according to the methodsof Zoller and Smith, supra, using the mutagenic oligonucleotide ZC1656(5' TGG GCC TCC GGC GTC CCC CTT 3' SEQ ID NO:3) and the "universal"second primer ZC87 (5' TCC CAG TCA CGA CGT 3' SEQ ID NO:4). Reactionproducts were screened using kinased ZC1656. Positive plaques werepicked, and template DNA was prepared and sequenced from the Pst I siteat 1077 to the Kpn I site at 1213. Sequence analysis confirmed thepresence of the desired mutation. The mutant clone was designated 1656.

An expression vector was then constructed using the 1656 clone. Themutagenized sequence was isolated from the M13 vector as a ˜0.14 kb PstI-Kpn I fragment. This fragment was ligated to the 1.7 kb Hind III-Xba Ifragment from FVII(565+2463)/pDX, the 0.5 kb Xba I-Pst I fragment fromFVII(565+2463)/pDX, and the 4.3 kb Kpn I-Hind III fragment fromFVII(565+2463)/pDX, as shown in the Figure. The presence of the desiredmutant sequence was confirmed by digesting mutant and wild-type cloneswith Pst I, and a mutant Factor VII insert in M13 with Kpn I and Xba I,preparing Southern blots of the digested DNA, and probing the blots withradiolabeled ZC1656.

The baby hamster kidney cell line BHK 570 (deposited with the AmericanType Culture Collection under accession number 10314) was transfectedwith two isolates (designated #544 and #545) of the 1656 expressionvector. The cells were prepared by diluting a confluent 10 cm plate ofBHK 570 cells 1:10 into five 10 cm plates in non-selective medium(Dulbecco's modified Eagle's medium [DMEM] containing 10% fetal bovineserum and 1% PSN antibiotic mix [GIBCO Life Technologies, Gaithersburg,Md.]). After 24 hours, when the cells had reached 20-30% confluency,they were co-transfected with one isolate of the expression vectorencoding the 1656 mutation, plasmid p486 (comprising the Adenovirus 5ori, SV40 enhancer, Adenovirus 2 major late promotor, Adenovirus 2tripartite leader, 5' and 3' splice sites, the DHFR^(r) cDNA and SV40polyadenylation signal in pML-1 (Lusky and Botchan, Nature 293: 79-81,(1981)) and 10 μg of carrier DNA (sonicated salmon sperm DNA) as shownin Table 1. The DNA was added to a 15 ml tube, then 0.5 ml of 2× Hepes(25 g Hepes, 40 g NaCl, 1.8 g KCl, 0.75 g Na₂ HPO₄.2H₂ O, 5 g dextrosediluted to 2.5 l with distilled water and pH adjusted to pH 6.95-7.0)was added and the tubes were mixed. The DNA in each tube wasprecipitated by the addition of 0.5 ml of 0.25 M CaCl₂ while air wasbubbled through the DNA/Hepes solution with a pasteur pipet. The tubeswere then vortexed, incubated at room temperature for 15 minutes, andvortexed again. The DNA mixtures were then added dropwise onto theplates of cells with a pipette. The plates were swirled and incubated at37° C. for 4-6 hours. After incubation, 2 ml of 20% glycerol diluted inTris-saline (0.375 g KCl, 0.71 g Na₂ HPO₄, 8.1 g NaCl, 3.0 g Tris-HCl,0.5 g sucrose, diluted in a total of 1 liter and pH adjusted to pH 7.9)was then added to each plate. The plates were swirled and left at roomtemperature for two minutes. The medium was then removed from the platesand replaced with 2 ml of Tris-saline. The plates were left at roomtemperature for 2 minutes, then the Tris-saline was removed and replacedwith 10 ml of non-selective medium. The plates were incubated at 37° C.for two days.

                  TABLE 1                                                         ______________________________________                                                Transfection*                                                         Plasmid Name                                                                            544    545      544 Control                                                                           545 Control                                 ______________________________________                                        Clone 544 15 μl                                                                             --       15 μ1                                                                              --                                          Clone 545 --      30 μ1                                                                              --      30 μ1                                    p486      1.5 μl                                                                            1.5 μ1                                                                              --      --                                          Carrier DNA                                                                             1.6 μl                                                                            1.6 μ1                                                                              1.6 μ1                                                                             1.6 μ1                                   ______________________________________                                         *DNA concentrations used were: clone 544, 0.7 μg/μl; clone 545, 0.3     μg/μl; p486, 1.49 μg/μl.                                     

After the two day incubation, the cells were diluted in selection medium(DMEM containing 10% dialyzed fetal bovine serum, 1% PSN antibiotic mixand 150 nM methotrexate) and 40 plated at dilutions of 1:100, 1:250 and1;500 in maxi plates.

The plates were incubated at 37° C. for one week. After one week, themedium was changed and replaced with selection medium, and the plateswere checked for colony formation.

Eight days later, after colony formation, twelve colonies were randomlychosen from the 1:500 dilution plates of the #544 and #545 transfectionplates. Each clone was plated into one well of a 6-well plate and grownin selection medium. After seven days, the plates were confluent, andthe clones were each split into 10 cm plates in selection medium.

The clones described above and control cells transfected to expresswild-type factor VII were metabolically labeled with ³⁵S-Methionine-Cysteine Protein Labeling Mix (NEN DuPont BiotechnologySystems, Wilmington, Del.). The clones were grown and prepared for apulse label experiment in selective medium. The cells were rinsed withphosphate buffered saline (Sigma, St. Louis, Mo.) and pulsed for fourhours in 20 μCi/ml ³⁵ S-Cys-³⁵ S-Met. After four hours, supernatants andcells were harvested. The cells were lysed essentially as described byLenk and Penman (Cell 16: 289-302, (1979)) and 400 μl of each lysate andprecleared with 50 pl of staph A (Sigma, St. Louis, Mo.).

Samples from the metabolically labeled cells wereradioimmunoprecipitated (RIP) by first incubating the samples with 6 μlof anti-Factor VII polyclonal antisera for four hours. Sixty microlitersof washed staphylococcal protein A was added to each sample, and thesamples were rocked for 1.5 hours at 40° C. The samples werecentrifuged, and the supernatant was removed. The pellets were washedtwice in 0.7 M RIPA buffer (10 mM Tris, pH 7.4, 1% deoxycholic acid[Calbiochem Corp., La Jolla, Calif.], 1% Triton X-100, 0.1% SDS, 5 mMEDTA, 0.7 M NaCl) and once in 0.15 M RIPA buffer (10 EM Tris, pH 7.4, 1%deoxycholic acid [Calbiochem Corp., La Jolla, Calif.], 1% Triton X-100,0.1 SDS, 5 mM EDTA, 0.15 M NaCl). One hundred microliters of 1× SDS dye(50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenolblue, 10% glycerol) was added to each sample, and the samples wereboiled for 5 minutes followed by centrifugation to remove the protein A.Fifty microliters of each sample was run on a 10% polyacrylamide gel.Results showed that 9 of 10 clones secreted modified Factor VII.

EXAMPLE II Anticoagulant Activity of Modified Factor VII

The ability of the modified Factor VII protein to inhibit clotting wasmeasured in a one-stage clotting assay using wild-type Factor VII as acontrol. Recombinant proteins were prepared essentially as describedabove from cells cultured in media containing 5μg/ml vitamin K. Varyingamounts of the modified Factor VII (from clone 544) or recombinantwild-type Factor VII were diluted in 50 mM Tris pH 7.5, 0.11% BSA to 100μl. The mixtures were incubated with 100 μl of Factor VII-deficientplasma (George King Bio-Medical Inc., Overland Park, Kans.) and 200 μlof thromboplastin C (Dade, Miami, Fla.; contains rabbit brainthromboplastin and 11.8 mM Ca⁺⁺). The clotting assay was performed in anautomatic coagulation timer (MLA Electra 800, Medical LaboratoryAutomation Inc., Pleasantville, N.Y.), and clotting times were convertedto units of Factor VII activity using a standard curve constructed with1:5 to 1:640 dilutions of normal pooled human plasma (assumed to containone unit per ml Factor VII activity; prepared by pooling citrated serumfrom healthy donors). Using this assay the preparations of modifiedFactor VII exhibited no detectable coagulant activity. Table 2 showsresults of the assay in terms of clotting times for control(untransfected) BHX cell-conditioned media (+/- vitamin K), wild-typeFactor VII and two isolates of cells expressing the modified Factor VII.Factor VII activity is seen as a reduction in clotting time over controlsamples.

                  TABLE 2                                                         ______________________________________                                        Sample         Dilution                                                                              Clotting Time (sec.)                                   ______________________________________                                        Control +K     1:5     33.1                                                                  1:10    33.4                                                   Control -K     1:5     34.3                                                                  1:10    33.2                                                   Wild-type      1:20    19.0                                                   Factor VII     1:40    21.5                                                                  1:80    23.3                                                   Modified       1:1     33.5                                                   Factor VII (#6)                                                               Modified       1:1     32.5                                                   Factor VII (#10)                                                              ______________________________________                                    

To determine the effect of the modified Factor VII on plasma factorsubstrates, preparations of modified Factor VII and recombinantwild-type or native Factor VII are incubated with either Factor X orFactor IX and the activation thereof monitored by clotting assays orpolyacrylamide gel electrophoresis.

EXAMPLE III Ability of Modified Factor VII to Bind Tissue Factor

The ability of the modified Factor VII to compete with wild-type FactorVII for tissue factor and inhibit its clotting activity was assessed ina one-step clotting assay in the presence of a limiting amount of tissuefactor (thromboplastin).

Clotting times were determined in a one-step assay similar to thatdescribed in Example II. A limited amount of tissue factor, a constantamount of wild type Factor VII, and increasing amounts of variant FactorVII were used in the mixing experiments. An inhibition of FactorVII/VIIa procoagulant activity would be seen as an increase in clottingtime in assays containing increasing amounts of variant Factor VII.

The amount of Factor VII activity in the test samples was calculated asa percentage of a standard curve that measured Factor VII activity innormal pooled plasma. The standard curve for Factor VII activity wasgenerated using serial dilutions of normal pooled plasma in phosphatebuffered solution (PBS) that ranged from 1:5 to 1:640. For this purposeit was assumed that normal plasma contains approximately 500 ng/ml ofFactor VII and this was considered to be one unit of activity. A mixtureof 100 μl Factor VII-deficient plasma, 100 μl plasma dilution and 200 μlof thromboplastin-C (Dade, Miami, Fla.) was used to measure clottingtime on a MLA Electra 800 automatic timer. To establish the standardcurve, the results were graphed as percentage of activity (1:5=100%activity) versus clotting time in seconds.

The assay required that the medium containing the wild type and variantFactor VII be composed of less than one percent serum. The dilutionswere made in PBS so that clotting times would fall along the standardcurve. A minimum dilution of 1:2 was typical. The final volume was 100μl. Two different human Factor VII Ser₃₄₄ →Ala variants, designatedclones "#10" and "#6" were tested in the experiments. The results, setforth in the Table below, show that as the amount of Factor VII variantincreased, the percent of Factor VIIa activity decreased.

                  TABLE 3                                                         ______________________________________                                        Results of mixing assay with Ser344 → Ala                              Variants (B4A1 (wild type) medium was used as 100%                            activity at 10 μl/reaction)                                                            Variant B4A1             Percent                                  Ser344 → Ala                                                                       medium  medium    BHK    FVIIa                                    Clone No.   amount  amount    Control*                                                                             Activity                                 ______________________________________                                        #10         10 μl                                                                              10 μl  0      70                                       #10         20 μl                                                                              10 μl  0      51                                       #10         30 μl                                                                              10 μl  0      43                                       #10         40 μl                                                                              10 μl  0      34                                       #10         50 μl                                                                              10 μl  0      28                                       #10 (-K).sup.§                                                                       20 μl                                                                              10 μl  0      78                                        #6         10 μl                                                                              10 μl  0      74                                        #6         20 μl                                                                              10 μl  0      56                                        #6         30 μl                                                                              10 μl  0      46                                        #6         40 μl                                                                              10 μl  0      41                                        #6         50 μl                                                                              10 μl  0      32                                        #6 (-K)    20 μl                                                                              10 μl  0      85                                       BHK control 0       10 μl  20 μl                                                                             91                                       BHK control (-K)                                                                          0       10 μl  20 μl                                                                             107                                      ______________________________________                                         *Untransfected conditioned medium                                             .sup.s For expression of the Factor VII variant, cells were grown in the      presence of vitamin K, except where noted "(-K)".                        

These experiments showed that variants of Factor VII having a Ser₃₄₄→Ala substitution competed with native Factor VII in a dose dependentfashion and inhibited the procoagulant activity of native FactorVII/VIIa. It can thus be concluded that Ser₃₄₄ →Ala variant human FactorVII competes with native human Factor VIIa and consequently inhibitsactivation of Factor X and/or IX in human plasma.

EXAMPLE IV Reaction of Factor VII with PPACK

Recombinant Factor VII was produced in transfected baby hamster kidneycells. The protein was purified and activated as disclosed by Thim etal. (Biochemistry 27: 7785-7793, 1988), Brinkous et al. (Proc. Natl.Acad. Sci. USA 86: 1382-1386, 1989) and Bjoern and Thim (Res. Discl. No.269, 564, 1986), which are incorporated herein by reference. The cellculture medium was recovered, filtered and diluted to reduce saltconcentration. The diluted medium was then fractionated by anionexchange chromatography using an elution buffer containing CaCl₂. TheFactor VII fraction was recovered and further purified byimmunochromatography using a calcium-dependent anti-Factor VIImonoclonal antibody. Additional purification was carried out using twoanion exchange chromatography steps wherein Factor VII was eluted usingCaCl₂ and NaCl, respectively. Factor VIIa was recovered in the finaleluate.

Recombinant Factor VIIa (1 μM) in 50 mM Tris-HCl, 100 mM NaCl, 5 mMCaCl2, ph 7.4 was incubated with 20 μM PPack(D-Phenylalanyl-Prolyl-Arginyl Chloromethyl Ketone; Calbiochem, LaJolla, Calif.) for 5, 20 and 60 minutes. Buffer containing thechromogenic substrate S2288 (H-D-Isoleucine-L-Prolyl-L-Argininep-nitroanilide; Kabi Vitrum AB, Molndal, Sweden) was then added toobtain a 2.5 fold dilution and a final concentration of 0.3 mM S2288.The generation of p-nitroaniline was measured and compared to resultsusing untreated Factor VIIa as a control. The results indicated thatFactor VIIa is fully inactivated after about 60 minutes under thesereaction conditions.

EXAMPLE V Generation of DEGR-Factor VIIa

Recombinant human Factor VIIa was prepared as described in Example IV.Recombinant human Factor VIIa, in 10 mM glycine buffer, pH 8.0, 10 mMCaCl₂, 50 mM NaCl, was diluted to a concentration of 1.5 mg/ml. A10-fold molar excess of Dansyl-L-Glu-Gly-Arg-Chloromethyl Ketone,DEGRck, (Calbiochem, La Jolla, Calif. 92037) which had been dissolvedwith distilled H₂ O was added to the Factor VIIa. After a 2 hrincubation at 37° C., a second 10-fold molar excess of DEGRck was addedto the mixture and incubated for an additional 2 hr at 37° C. A third10-fold molar excess of DEGRck was added to the Factor VIIa andincubated for approximately 16 hours at 4° C. The DEGR-Factor VIIasample was then extensively dialyzed at 4° C. against Tris bufferedsaline (0.05 M Tris-HCl, 0.1 M NaCl, pH 7.5) to remove any free DEGRck.

The final DEGR-Factor VIIa mixture was tested for the presence of freeDEGRck in a Factor Xa chromogenic substrate assay. The DEGR-Factor VIIamixture was added to purified human Factor Xa along with the chromogenicsubstrate S-2222. This substrate is cleaved specifically by Factor Xaand not by Factor VIIa. Unbound DEGRck in the mixture is able to bind tothe Factor Xa and there by inhibit the chromogenic activity of theFactor Xa. Spiking free DEGR-ck into a Factor Xa mixture generated astandard curve to measure the level of free DEGRck in solution versusthe inhibition of Factor Xa chromogenic activity. Analysis of theDEGR-Factor VIIa mixture showed that the ratio of freeDEGRck:DEGR-Factor VIIa was less than 0.5% following extensive dialysis,thereby ensuring that the inhibition observed by DEGR-Factor VIIa in thevarious assay systems described below was not due to the presence offree DEGRck.

EXAMPLE VI Factor Xa Generation on Rat Smooth Muscle Cells

Vascular smooth muscle cells were analyzed for the presence ofcell-surface tissue factor by measuring the ability of the cells tostimulate the conversion of Factor X to Factor Xa using a chromogenicsubstrate that is specific for Factor Xa.

Rat vascular smooth muscle cells (Clowes et al., J. Clin. Invest.93:644-651 (1994)) were plated into 96-well culture dishes (AmericanScientific Products, Chicago, Ill.) at 8,000 cells per well in growthmedia (Table 4).

                  TABLE 4                                                         ______________________________________                                        500  ml Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO-BRL,                      Gaithersburg, MD.)                                                       10%  fetal calf serum (Hyclone, Logan, UT.)                                   1    mM sodium pyruvate (Irvine, Santa Ana, CA.)                              0.29 mg/ml L-glutamine (Hazelton, Lenexa, KS.)                                1x   PSN; (100X is 5 mg/ml penicillin, 5 mg/ml streptomycin,                       10 mg/ml neomycin) (GIBCO-BRL, Gaithersburg, MD.)                        ______________________________________                                    

After a 48 hour incubation at 37° C. the medium was changed to serumfree medium (Table 5).

                  TABLE 5                                                         ______________________________________                                        250  ml Dulbecco's Modified Eagle's Medium (DMEM)                             250  ml Ham's F-12 Medium (Fred Hutchinson Cancer Research                         Center, Seattle, WA)                                                     1    mM sodium pyruvate                                                       .29  mg/ml L-glutamine                                                        20   μM transferrin (JRH, Lenexa, KS.)                                     5    μM insulin (GIBCO-BRL)                                                16   ng selenium (Aldrich, Milwaukee, WI.)                                    1    mg/ml bovine serum albumin (Sigma, St. Louis, MO)                        ______________________________________                                    

The cells were incubated 72 hours at 37° C. After incubation, eitherPDGF-BB (10 ng/ml) or 10% fetal calf serum was added to the cells tostimulate tissue factor expression (Taubman et al., J. Clin. Invest.21:547-552, 1993). A parallel set of cells received neither PDGF norserum to monitor for intrinsic activity of unstimulated cells. After a 6hour incubation, recombinant human Factor VIa was added to the cells ata final concentration of 10 nM. One set of cells did not have FactorVIIa added as a negative control. The cells were incubated for 2 hoursat 37° C. and washed with HEPES buffer (10 mM HEPES, 137 mM NaCl, 4 mMKCl, 5 mM CaCl₂, 11 mM glucose, 0.1% BSA). After washing, cells wereincubated for 5 min with 50 μl per well of 200 nM plasma-purified humanFactor X in a Tris-buffered saline supplemented with 5 mM CaCl₂.Twenty-five microliters of 0.5 M EDTA and 25 μl of an 800 μM solution ofS-2222 chromogenic substrate (Kabi Pharmacia, Franklin, Ohio.) wereadded to each well. The plates were incubated for 40 min at roomtemperature, then analyzed at 405 nm using a THERMOMAX microplate reader(Molecular Devices, Menlo Park, Calif.).

Table 6 shows an increase in absorbance for the Factor VIIa treatedwells as compared to the control wells (no Factor VIIa added). Theincrease in absorbance is a direct measurement of the level of Factor Xagenerated in the wells and its subsequent cleavage of the chromogenicsubstrate, releasing the chromophore. The data also demonstrate that thelevel of chromogenic activity in cells pretreated with either PDGF-BB or10% fetal calf serum was higher than unstimulated cells.

                  TABLE 6                                                         ______________________________________                                               Test Sample                                                                           OD.sub.405                                                     ______________________________________                                               Control .043                                                                  Intrinsic                                                                             .247                                                                  PDGF-BB .360                                                                  10% FCS .342                                                           ______________________________________                                    

These results clearly show there is a Factor VIIa-dependent activationof Factor X to Factor Xa on the cell surface of rat vascular smoothmuscle cells.

EXAMPLE VII Inhibition of Cell-Surface Chromogenic Activity ByDEGR-Factor VIIa

Rat vascular smooth muscle cells were plated into 96-well culture dishesas described above. The cells were cultured for 72 hours in serum freemedia as described above and treated with the addition of 10% fetal calfserum for 6 hours to stimulate tissue factor expression. Afterstimulation, buffer only (control), 10 nM Factor VIIa, or 10 nM FactorVIIa+100 nM DEGR-Factor VIIa was added to each well. The cells wereincubated for 2 hours at 37° C., then washed with HEPES buffer. Afterwashing, the cells were incubated for 5 minutes with 50 μl per well of200 nM Factor X in Tris-buffered saline supplemented with 5 mM CaCl₂.Twenty-five microliters of 0.5 M EDTA and 25 μl of S-2222 (800 μM)chromogenic substrate (Kabi Pharmacia) were added to each well. Thecells were incubated at room temperature for 40 minutes. Chromogenicactivity was analyzed at 405 nm as described above.

Table 7 shows stimulation of chromogenic activity in the wells treatedwith Factor VIIa only, and inhibition of stimulation when DEGR-FactorVIIa was co-incubated with the Factor VIIa. These results demonstratethat DEGR-Factor VIIa acts as a competitive antagonist for Factor VIIabinding, thereby inhibiting the activation of Factor X to Factor Xa andthe subsequent cleavage of the S-2222 chromogen.

                  TABLE 7                                                         ______________________________________                                        Test Sample          OD.sub.405                                               ______________________________________                                        Control              .035                                                     Factor VIIa          .342                                                     Factor VIIa + DEGR-Factor VIIa                                                                     .073                                                     ______________________________________                                    

EXAMPLE VIII Dose Dependent Inhibition by DEGR-Factor VIIa of CellSurface Chromogenic Activity on Rat Smooth Muscle Cells

Rat vascular smooth muscle cells were plated into 96-well culture dishesat 4,000 cells per well in growth medium supplemented with 1% fetal calfserum (as in Table 4 without 10% fetal calf serum). After 5 days themedium was removed, and either increasing concentrations of Factor VIIaalone or 10 nM Factor VIIa with increasing concentrations of DEGR-FactorVIIa were added to the cells. The cells were incubated with the FactorVII mixtures for 2 hours at 37° C. After incubation, the cells werewashed and incubated with 50 μl of 200 nM Factor X in tris bufferedsaline for 5 minutes at room temperature. Each well had 25 μl of 0.5 MEDTA and 25 μl of 800 μM S-2222 (Kabi Pharmacia) added to it, and theplates were incubated for 40 minutes at room temperature. Chromogenicactivity was analyzed at 405 nm in a microplate reader as describedabove.

Table 8 shows a dose-dependent increase in chromogenic activity withincreasing amounts of Factor VIIa added to the wells. When the mixtureof DEGR-Factor VIIa with 100 nM Factor VIIa was added to the cells(Table 9) there was a dose dependent inhibition in chromogenic activity.A 1:1 molar ratio of DEGR-Factor VIIa:Factor VIIa inhibitedapproximately 95% of the chromogenic activity. These data suggest thatin this experimental design DEGR-Factor VIIa has a significantly higheraffinity for cell-surface tissue factor than native Factor VIIa onsmooth muscle cells in culture. If DEGR-Factor VIIa and Factor VIIa hadequal affinity for binding tissue factor then the level of inhibitionobserved when the two molecules were added to the cells in an equalmolar ratio would not have been as high.

                  TABLE 8                                                         ______________________________________                                               Factor VIIa                                                                   Conc. (nM)                                                                            OD.sub.405                                                     ______________________________________                                               .10     .005                                                                  .39     .025                                                                  1.56    .058                                                                  6.25    .111                                                                  25.00   .154                                                                  100.00  .208                                                           ______________________________________                                    

Table 9 shows the dose dependent inhibition of Factor Xa chromogenicactivity on rat smooth muscle cells by DEGR-Factor VIIa. Increasingconcentrations of DEGR-Factor VIIa were co-incubated with 100 nM FactorVIIa, and the Factor Xa chromogenic activity determined usingchromogenic substrate S-2222.

                  TABLE 9                                                         ______________________________________                                               DEGR-Factor                                                                   VIIa Conc. (nM)                                                                         OD.sub.405                                                   ______________________________________                                               .00       .208                                                                .39       .176                                                                1.56      .116                                                                6.25      .073                                                                25.00     .026                                                                100.00    .014                                                         ______________________________________                                    

EXAMPLE IX Inhibition of Factor Xa generation by DEGR-Factor VIIa in asoluble tissue factor assay.

The conversion of Factor X to Factor Xa using purified recombinantsoluble tissue factor was established using a chromogenic assay. Tissuefactor was expressed and purified from Saccharomyces cerevisiae(Shigematsu et al., J. Biol. Chem. 267:21329-21337, 1992). Solubletissue factor was purified and characterized by Dr. W. Kisiel(University of New Mexico). A reaction mixture containing 65.9 μl ofsoluble tissue factor (2.2 μM), 29.0 μl of PCPS (1 mM, Sigma, St. Louis,Mo.), 29.5 μl human Factor X (4.1 μM), 2.77 ml Hank's buffer (25 mMTris, pH 7.4, 150 mM NaCl, 2.7 mM KCl, 5 ]nM CaCl₂, 0.1% BSA) wasprepared. Forty microliter of tissue factor/Factor X mixture, 25 μlFactor VIIa diluted with TBS and 25 μl of DEGR-Factor VIIa diluted withTBS were added to each well of a 96-well microtiter plate. A controlusing 40 p1 of tissue factor/Factor X mixture; 25 μl Factor VIIa dilutedwith TBS, and 25 μl of TBS only was included. Ten microliters of S-2222(4 mM) chromogenic substrate was added to the reaction mixture in thewells and incubated at room temperature for 2-10 minutes. Results wereanalyzed at 405 nm in a microplate reader as described above.

Determination of a standard curve for Factor VIIa activation of Factor Xwas made using increasing concentrations of Factor VIIa added in theabsence of DEGR-Factor VIIa. The results, presented in Table 10, showthat there is a dose-dependent increase in chromogenic activity withincreasing amounts of Factor VIIa added to the reaction mixture. Thesimultaneous addition of varying amounts of DEGR-Factor VIIa and 100 nMFactor VIIa led to a dose dependent decrease in chromogenic activity(Table 11). These data demonstrate that DEGR-Factor VIIa acts as acompetitive antagonist for native Factor VIIa binding to soluble tissuefactor, and thereby inhibits the generation of Factor Xa as measured bythe decrease in chromogenic activity towards the chromogenic substrateS-2222.

                  TABLE 10                                                        ______________________________________                                        Stimulation of Factor Xa chromogenic activity with                            increasing concentrations of Factor VIIa added to soluble                     tissue factor. Changes in optical density were measured using                 chromogenic substrate S-2222.                                                        Factor VIIa                                                                   Conc (nM)                                                                             OD.sub.405                                                     ______________________________________                                               .78     .168                                                                  1.56    .288                                                                  3.12    .478                                                                  6.25    .694                                                                  12.50   .764                                                                  25.00   .790                                                                  50.00   .738                                                                  100.00  .770                                                           ______________________________________                                    

                  TABLE 11                                                        ______________________________________                                        Inhibition of Factor Xa chromogenic activity by the addition                  of DEGR-Factor VIIa to soluble tissue factor in the                           presence of native Factor VIIa is measured. Changes in optical                density were measured using the chromogenic substrate S-2222.                        DEGR-Factor                                                                   VIIa Conc. (nM)                                                                         OD.sub.405                                                   ______________________________________                                                0        .810                                                                 50       .750                                                                100       .609                                                                200       .296                                                                400       .167                                                                800       .083                                                                1600      .055                                                         ______________________________________                                    

EXAMPLE X Inhibition of Coagulation by DEGR-Factor VIIa

Standard clotting assays to monitor the effect of DEGR-Factor VIIa onclotting time were prepared as follows: 100 μl of normal baboon plasma,collected with sodium citrate as anticoagulant, was added to 100 μl ofvarying concentrations of DEGR-Factor VIIa diluted in TBS (20 mM Tris,pH 7.4, 150 mM NaCl). The samples were mixed and briefly incubated at37° C. The samples were added to an Electra 800 Automatic CoagulationTimer (Medical Laboratories Automation, Pleasantville, N.Y.). Afterincubation, 200 μl of a tissue factor preparation containing 25 mM CaCl₂was added to the DEGR-Factor VIIa preparations. A tissue factorpreparation was made as a saline extract of baboon brain from freshlyfrozen brain tissue and characterized for its ability to initiatecoagulation in baboon plasma. A concentration of tissue factor that gavea clotting time of about 40 seconds was selected.

The data, presinted in Table 12, demonstrates a dose-dependent increasein clotting time due to the addition of DEGR-Factor VIIa. A dose as lowas 1 μg/ml of DEGR-Factor VIIa in plasma resulted in a significantincrease in clotting time.

                  TABLE 12                                                        ______________________________________                                        Dose dependent increase in clotting time due to                               DEGR-Factor VIIa.                                                             DEGR-Factor VIIa                                                                              Clotting time                                                 (μg/ml plasma)                                                                             (seconds)                                                     ______________________________________                                        0               40.7                                                          0.5             46.2                                                          1.0             50.8                                                          2.5             64.5                                                          5.0             108.1                                                         10.0            158.4                                                         ______________________________________                                    

EXAMPLE XI Inhibition of Platelet Accumulation With DEGR-Factor VIIa

DEGR-Factor VIIa was analyzed for its ability to inhibit plateletaccumulation at sites of arterial thrombosis due to mechanical injury innon-human primates. A model of aortic endarterectomy was utilized inbaboons, essentially as described by Lumsden et al. (Blood 81:1762-1770(1993)). A section of baboon aorta 1-2 cm in length was removed,inverted and scraped to remove the intima of the artery andapproximately 50% of the media. The artery was reverted back to itscorrect orientation, cannulated on both ends and placed into anextracorporeal shunt in a baboon, thereby exposing the mechanicallyinjured artery to baboon blood via the shunt. Just prior to opening ofthe shunt to the circulating blood, ¹¹¹ In-labeled autologous plateletswere injected intravenously into the animal. The level of plateletaccumulation at the site of the injured artery was determined byreal-time gamma camera imaging.

Evaluation of DEGR-Factor VIIa for inhibition of platelet accumulationwas done using bolus injections of DEGR-Factor VIIa or saline controland were given just prior to the opening of the shunt. The injuredarteries were measured continuously for 60 minutes. A dose of 0.005mg/kg of DEGR-Factor VIIa inhibited platelet accumulation. At a 1.0mg/kg bolus injection, approximately 90% of platelet accumulation wasinhibited at 1 hour post drug administration. These results are shown inFIG. 2.

These data show that inhibition of tissue factor with DEGR-Factor VIIacan significantly inhibit the development of platelet-rich thrombi in anonhuman primate model of acute vascular injury.

EXAMPLE XII DEGR-FVIIa Inhibits Vascular Restenosis Following BalloonAngioplasty in Atherosclerotic Rabbits

DEGR-FVIIa was evaluated for its ability to modulate lesion developmentfollowing balloon angioplasty in New Zealand White (NZW) atheroscleroticrabbits. This animal model has been well characterized and has proven tobe a good model for evaluating anti-thrombotic compounds on vascularlesion development (Gimple et al., Circulation 86:1536-1546 (1992), andRogosta et al., Circulation 89:1262-1271 (1994)). The animal model usedto evaluate DEGR-FVIIa is essentially as described by Ragosta, ibid.

Anesthesia was induced in rabbits with 5 mg/kg xylazine and 35 mg/kgketamine by intramuscular injection. The proximal femoral arteries wereexposed by cutdown below the inguinal ligament with proximal and distalligatures. The isolated segments were cannulated with 27 gauge needles.A vent was created by needle puncture. The isolated segments wereflushed with saline to clear residual blood, and desiccated by airinfused at a rate of 80 ml/min for 8 minutes. Following air-drying, theisolated segments were again flushed with saline and the ligaturesremoved. Hemostasis was maintained with non-occlusive local pressure.The segments were demarcated with metal clips. Local spasm was treatedwith Xylocaine 1% locally. The day following surgery, the animals wereplaced on 1% cholesterol and 6% peanut oil diet for one month untilballoon angioplasty. Tylenol 10 mg/kg orally was given for postoperativepain relief for 3-5 days. Ambipen 1cc was given after the surgicalprocedure during postoperative days 3 to 5.

The test drug delivery for the animals consisted of an initial bolusinjection immediately prior to balloon angioplasty, followed by acontinuous systemic infusion by osmotic pump via the internal jugularvein. The duration of the drug infusion was 3 days. The control animalsreceived heparin, 150 U/kg IV bolus, prior to balloon angioplastyfollowed by saline infusion. The DEGR-FVIIa treated animals received a 1mg/kg bolus injection followed by 50 μg/kg/hr infusion.

For the placement of the osmotic pumps for continuous systemic infusion,anesthesia was induced in the animals as described above, and maintainedthroughout the procedure with additional IM injections of ketamine andXylazine. Through a midline neck incision, the right internal jugularvein was isolated by blunt dissection and the distal end ligated. Asilastic tube (PE-160) was introduced into the right internal Jugularvein. A subcutaneous tunnel was created to pass the silastic tube. Thistube was connected with the osmotic pump. The osmotic pump was implantedsubcutaneously in the back of the rabbit. The right common carotidartery was isolated by blunt dissection and the distal end ligated. Viaan arteriotomy, a 5F introducer was placed and advanced to the junctionof the aortic arch. Blood was drawn for determination of hemostaticparameters, drugs and cholesterol levels. Twenty milligrams of xylocainwas injected intraarterially. A control aortoiliofemoral angiogram wasperformed via a 5F Berman catheter positioned above the aorticbifurcation using 3-4 ml renographin injected over 3 seconds by hand.

After removal of the Berman catheter, a 0.014-inch guidewire wasintroduced in the descending aorta and positioned above the aorticbifurcation. Under fluoroscopic guidance, an appropriately sized balloonangioplasty catheter of 2.0 to 2.5 mm was introduced and advanced overthe guidewire and positioned across the stenosis. The balloon wasinflated to 6 atmospheres for 60 seconds with a hand inflator. Threeinflations were performed with 60 second intervals between inflations.This procedure was performed in both femoral arteries in each animal.

Following balloon dilatation, the angioplasty catheter was withdrawn andthe Berman catheter reintroduced to a position 3 cm above the aorticbifurcation. To minimize spasm 20 mg of lidocaine was givenintraarterially. A post procedure angiogram was performed as describedabove. A 1 cm grid was positioned at the level of the femoral artery tocalculate the actual diameter. The catheter was then removed. The rightcarotid artery was ligated with 3-0 silk and the wound sutured bylayers. Ambipen and acetaminophen were given as above.

Prothrombin time and concentration of DEGR-FVIIa in the blood weredetermined at immediately pre-bolus injection of the test compound, 1 hrpost bolus injection, and at 3 days at the end of continuous infusion.One to two mls of citrated plasma was obtained and the prothrombin timesand antigen levels determined.

A standard clotting assay was used to monitor the prothrombin time inthe control and DEGR-FVIIa-treated animals as follows. Twenty-fivemicroliters of test rabbit plasma, collected with sodium citrate asanticoagulant, was added to 150 μl of TBS (20 mM Tris, pH 7.4, 150 mMNaCl). The samples were mixed and added to an Electra 800 AutomatedCoagulation Timer (Medical Laboratories Automation, Pleasantville,N.Y.). After incubation, 200 μl of thromboplastin preparation (SigmaChemical) containing 25 mM CaCl₂ was added to the plasma preparations. Aconcentration of thromboplastin that gave a clotting time ofapproximately 20 seconds in the control rabbit plasma was selected.

An ELISA assay was used to determine the concentration of DEGR-FVIIa inplasma samples from the control and DEGR-FVIIa treated rabbits. Theassay involved first diluting an anti-human FVII monoclonal antibody(Dr. W. Kisiel, U. of New Mexico) to 2.0 μg/ml in 0.1 M carbonate bufferpH 9.6, and adding 100 μl/well to 96-well plates. The plates were thenincubated at 4° C. overnight and subsequently washed two times usingwash buffer (PBS, pH 7.4, containing 0.05% Tween 20). Blocking ofnonspecific binding sites was achieved with 200 μl of blocking bufferper well (PBS, pH 7.4, containing 0.05% Tween 20 and 1% BSA) incubatedat 37° C. for 2 hr, followed by a wash using the wash buffer.

After blocking, a standard dilution series of DEGR-FVIIa ranging from20-0.027 ng/ml was added, along with a dilution series of the testrabbit plasma (1:100 to 1:4000 in blocking buffer) applied at 100μl/well. Non-immune rabbit plasma was used as a negative control. Plateswere then incubated for 1 hr at 37° C. followed by four washes with washbuffer.

DEGR-FVIIa was detected by adding 100 μl/well of a 1:1,000 dilution ofrabbit anti-human FVII polyclonal antibody (Dr. Kisiel, U. New Mexico)in blocking buffer. Plates were incubated for 1 hr at 37° C., followedby five washes with wash buffer. Specific antibody binding was detectedusing 100 μl/well of a 1:2,000 dilution of goat anti-rabbit IgGantibody-peroxidase conjugate (Tago, Inc.). Plates were incubated for 1hr at 37° C. and washed six times with wash buffer. Finally, 100 μl ofsubstrate solution was added (0.42 mg/ml of o-phenylenediaminedihydrochloride [OPD] in 0.2 M citrate buffer, pH 5.0, containing 0.3%H₂ O₂). After 1-3 min at room temp. the color reaction was stopped byadding 100 μl/well of 1N H₂ SO₄ and the plates were read at 490 nm on aMicroplate spectrophotometer. The concentration of DEGR-FVIIa in theplasma samples was determined by comparing the A₄₉₀ values of theunknown to those of the DEGR-FVIIa standard curve.

Analysis of plasma samples for prothrombin times and DEGR-FVIIa antigenlevels is shown in Table 13 and Table 14, respectively. The data arepresented for each individual animal. Table 15 shows a summary of themean clotting times. In all cases, the DEGR-FVIIa treated animals hadelevated prothrombin times at the 1 hr post-bolus injection time pointwhich returned to near pre-treatment levels at the 3-day time point.Analysis of the DEGR-FVIIa antigen levels also showed a high level ofDEGR-FVIIa in the plasma at the 1 hr time point, ranging between 2-6μg/ml in the plasma, with much lower circulating levels at the 3 daytime point. The levels of DEGR-FVIIa measured at the 1 hr time periodcorrespond with a predicted increase in prothrombin time, as determinedby spiking normal rabbit plasma with DEGR-FVIIa in vitro and determiningprothrombin times in a standard dilute thromboplastin assay.

                  TABLE 13                                                        ______________________________________                                        MEASUREMENT OF PROTHROMBIN TIMES                                              Animal            Clotting Time (seconds)                                     Number Treatment  Pretreatment                                                                              1 hour                                                                              3 days                                    ______________________________________                                        73     Control    24.8        22.3  17.8                                      74     Control    24.8        27.9  18.6                                      75     Control    24.6        N/D   20.5                                      76     Control    22          N/D   17.9                                      169    Control    21.2        22.9  22                                        170    Control    24.9        23.5  18.6                                      173    Control    25.9        21    20.8                                      174    Control    25          29.4  20.1                                      77     DEGR-FVIIa 22.5        40.1  18.3                                      78     DEGR-FVIIa 24.3        34    20.9                                      80     DEGR-FVIIa 24.7        50    21.7                                      96     DEGR-FVIIa N/A         N/A   21                                        97     DEGR-FVIIa 23.6        33.3  21.2                                      171    DEGR-FVIIa 20.6        45.8  21.9                                      172    DEGR-FVIIa 23.5        41.6  22.4                                      ______________________________________                                         N/A  Data Not Available                                                  

                  TABLE 14                                                        ______________________________________                                        ELISA TO DETECT DEGR-FVIIa IN RABBIT PLASMA                                                   FVIIa ELISA (ng/ml)                                           Animal Number                                                                           Treatment   Pretreatment                                                                             1 hour                                                                              3 days                                 ______________________________________                                         73       Control      0         13     0                                      74       Control     36         14     4                                      75       Control      0         N/A    9                                      76       Control      0         N/A    14                                    169       Control      0         0      1                                     170       Control      0         0      0                                     173       Control     36         31     0                                     174       Control     87         86    160                                     77       DEGR-FVIIa   0         3,210 102                                     78       DEGR-FVIIa   1         4,950  7                                      80       DEGR-FVIIa  13         4,543 661                                     96       DEGR-FVIIa  65         4,900 117                                     97       DEGR-FVIIa   4         4,600 502                                    171       DEGR-FVIIa  13         2,145 212                                    172       DEGR-FVIIa   9         2,830 228                                    ______________________________________                                         N/A = Data Not Available                                                 

                  TABLE 15                                                        ______________________________________                                        Statistical Summary of Plasma Clotting Times.                                 ______________________________________                                        Unpaired t-Test X          PRE-BLEED                                          DF: 12      Unpaired t Value: 1.12                                                                       Prob. (2-tail): .2852                              Group:     Count:  Mean:     Std. Dev.:                                                                           Std. Error                                ______________________________________                                        Control    8       24.15     1.64    .58                                      DEGR-VIIa  6       23.2      1.48   .6                                        ______________________________________                                        Unpaired t-Test X          1 Hr POST ANGIO                                    DF: 10      Unpaired t Value: -5.44                                                                      Prob. (2-tail): .0003                              Group:     Count:  Mean:     Std. Dev.:                                                                           Std. Error                                ______________________________________                                        Control    6       24.5      3.35   1.37                                      DEGR-VIIa  6       40.8      6.53   2.67                                      ______________________________________                                        Unpaired t-Test X                                                                         3 Days POST ANGIO                                                 DF: 13      Prob. (2-tail): .0622                                             Unpaired t Value: -2.04                                                       Group      Count   Mean      Std. Dev.                                                                            Std. Error                                ______________________________________                                        Control    8       19.54     1.53    .54                                      DEGR-VIIa  7       21.06     1.33   .5                                        ______________________________________                                    

Three weeks post-angioplasty a follow-up angiogram was repeated asdescribed above via the left carotid artery immediately prior tosacrifice. Through a vertical lower abdominal incision, the distal aortawas isolated, tied off proximally, and the perfusion cannula insertedabove the aortic bifurcation. The distal aorta was flushed with 50 ml ofsaline followed by in vivo fixation with 500 ml of Histochoice (AMRESCO,Solon, Ohio.) solution infused over 15 mins at 120 mmHg. Once perfusionwas started, the animals were sacrificed with an overdose of nembutal (3ml sodium pentobarbital IV, 65 mg/ml). A 5 cm segment of femoral arterywas excised bilaterally. The tissue was preserved in Histochoicesolution for light microscopy.

To determine intimal lesion development at the site of balloonangioplasty, the excised femoral arteries were cut in serial 3 mmsections, embedded in paraffin, and sections cut from multiple regionsof each artery. The sections were mounted onto glass slides and theslides stained with hematoxylin and eosin, and Van Giemson stains.Morphometric analysis was performed with Bioquant Program to obtain areameasurements for the lumen, the intima and the media. Morphometricanalysis of tissue sections from the injured arteries were donemeasuring the total luminal area; the area of the intima, determined bymeasuring the area within the internal elastic lamina and subtractingthe corresponding luminal area from each tissue section; and the area ofthe media, determined by measuring the area inside the external elasticlamina and subtracting the area inside the internal elastic lamina.Measurements for intimal lesions in the femoral arteries in control andDEGR-FVIIa treated animals showed that there was a significant decreasein the size of the intima in the DEGR-FVIIa treated animals (Table 16).In contrast, measurement of the medial area showed no significantdifference between the two groups.

                  TABLE 16                                                        ______________________________________                                        MEASUREMENTS OF THE INTIMA AND MEDIA IN                                       BALLOON ANGIOPLASTY TREATED RABBITS                                           Group     N                 Std. Dev.                                                                            Prob. (2-tail)                             ______________________________________                                                        Intima (mm2)                                                  Control   13    0.819       0.414  0.0138                                     DEGR-FVIIa                                                                              10    0.438       0.192                                                             Media (mm2)                                                   Control   13    0.389       0.098  0.172                                      DEGR-FVIIa                                                                              10    0.329       0.105                                             ______________________________________                                    

The data from the angiographic measurements are presented in Table 17 asthe Mean Luminal Diameter (MLD) +/-standard deviation for the controland DEGR-FVIIa treated animal for all three time points: immediatelypre-angioplasty, immediately post-angioplasty, and 21 dayspost-angioplasty. There was no significant difference in the MLD betweenthe control and DEGR-FVIIa treated animals at either the pre- orimmediately post-angioplasty measurements. A significant increase in MLDwas observed, however, in the DEGR-FVIIa treated animals at the 21 daypost-angioplasty measurement.

                  TABLE 17                                                        ______________________________________                                        MEASUREMENT OF MINIMAL LUMINAL DIAMETER (MLD)                                 Group     N     Meal MLD    Std. Dev.                                                                            Prob. (2-tail)                             ______________________________________                                        Pre-PTCA Measurement of MLD                                                   Control   13    1.202       0.24   0.3883                                     DEGR-FVITa                                                                              10    1.283       0.19                                              Post-PTCA Measurement of MLD                                                  Control   13    1.492       0.551  0.5326                                     DEGR-FVIIa                                                                              10    1.323       0.725                                             21 Day Measurement of MLD                                                     Control   13    0.889       0.228  0.0001                                     DEGR-FVIIa                                                                              10    1.393       0.242                                             ______________________________________                                    

EXAMPLE XIII

Inhibition of Cell-Surface Factor Xa Generation on Baboon SMCs byDEGR-FVIIa

A cell-surface chromogenic assay was developed, essentially as describedin Example VIII above, to measure the efficacy of DEGR-FVIIa to blockFVIIa binding to cell-surface tissue factor and the subsequentconversion of Factor X to Factor Xa on monolayers of baboon smoothmuscle cells (SMCs). This method is a modification of those described bySakai et al., J. Bio. Chem. 264:9980-9988 (1989) and Wildgoose et al.,Proc. Natl. Acad. Sci. USA. 87:7290-7294 (1990). Baboon SMCs wereobtained from the University of Washington, Seattle, Wash., and werecultured from aortic explants. The baboon SMCs were plated into 96-wellculture dishes at a concentration of 8,000 cells/well in 200 μl/wellDMEM culture media supplemented with 10% fetal calf serum, andmaintained in this media for 4 days at 37° C. in 5% CO₂. At the time ofassay 110 μl of culture media was removed, and increasing concentrationsof FVIIa or FVIIa in combination with DEGR-FVIIa were added to wells. Astandard curve for FVIIa concentration was generated, ranging from 5 nMto 0.04 nM. To measure the inhibitory activity of DEGR-FVIIa on FVIIaactivity, increasing concentrations of DEGR-FVIIa were added to testwells in the presence of a constant amount of FVIIa (5 nM). Both FVIIaand DEGR-FVIIa were diluted with HEPES buffer (10 mM HEPES, 137 mM NaCl,4 mM KCl, 5 mM CaCl₂, 11 mM glucose, 0.1% BSA) and 10 μl of 10× stocksolutions added to the cells. The cells were incubated with the testcompounds for 2 hr at 37° C., then washed 3 times with HEPES buffer.Fifty microliters of a 200 nM solution of Factor X in Tris buffer (25 mMTris, pH 7.4, 150 mM NaCl, 2.7 mM KCl, 5 mM CaCl₂, 0.1 BSA) was thenadded to each well. After 4 mins at room temp., 25 μl of 0.5 M EDTA wasadded to stop the Factor X to Xa conversion. Twenty-five microliters perwell of 0.8 mM S-2222, a factor Xa-specific chromogenic substrate, inTris buffer was added and the absorbance at 405 nM read after 60 mins ina Thermomax microplate reader (Molecular Devices Corp., Menlo Park,Calif.).

The results, shown in FIG. 3, demonstrate a dose dependent increase inamidolytic activity for the FVIIa treated wells (open boxes). Theincrease in absorbance is a direct measure of the level of Factor Xagenerated in the wells and its subsequent cleavage of the chromogenicsubstrate. The addition of increasing amounts of DEGR-FVIIa with aconstant amount of FVIIa (5 nM) showed a dose dependent decrease inamidolytic activity with increasing levels of DEGR-FVIIa (closed boxes).An equal molar ratio of DEGR-FVIIa to FVIIa was able to inhibit >90% ofthe chromogenic activity. Even at a 10-fold lower level of DECR-FVIIa,there was still a 40% inhibition in the generation of Factor Xachromogenic activity. These results support the conclusion thatDEGR-FVIIa is an extremely potent antagonist of the activation of FactorX to Xa by FVIIa on the surface of intact cell monolayers of SMCs.

EXAMPLE XIV Effect of DEGR-Factor VIIa on Vascular Thrombosis Formationand Vascular Lesion Formation in Baboons

Human DEGR-Factor VIIa was tested for the ability to inhibit tissuefactor (TF) and activated Factor VII (FVIIa) mediation of vascularlesion formation (VLF) induced by mechanical vascular injury in nonhumanprimates.

Beginning immediately prior to creating mechanical vascular injury inbaboons, DEGR-Factor VIIa was infused intravenously for 7 days (5animals) or 30 days (1 animal). Measurements were performed for vascularlesion formation on day 30. The results in 5 treated animals werecompared with the findings in 5 concurrent vehicle buffer-infusedcontrols.

Baseline measurements were obtained on study animals for: a) plateletcounts, neutrophil counts, monocyte counts and red cell counts; b)plasma fibrinogen level; c) activity levels of plasma coagulationfactors VII, VIIa, X and V, together with the antigenic levels of FVII;and d) baseline plasma sample for anti-Factor VIIa antibody level.

Under halothane anesthesia and sterile operating conditions, animalslabeled with autologous ¹¹¹ In-platelets received intravenous infusionsof DEGR-FVIIa using the tether system for continuous intravenousadministration (initial bolus injection of 1 mg/kg followed bycontinuous intravenous infusion of 50 μg/kg/hr. The animals receivedsurgical carotid endarterectomy, bilateral brachial artery or bilateralfemoral artery Fogarty balloon catheter angioplasties.

The DEGR-FVIIa was administered for 7 or 30 days by continuous infusionsvia venous catheter using the tether system. Thirty days after surgerythe animals were anesthetized with halothane and underwent in situpressure-perfusion fixation with 4% paraformaldehyde containing 0.1%glutaraldeyde for 30 min. At that time, vascular segments (containingthe sites previously injured) were harvested using procedures of Harkeret al., Circulation 83:41-44 (1991) and Hanson et al., Hypertension18:I1170-I176 (1991). The specimens were post-fixed in vitro (4%paraformaldehyde containing 0.1% glutaraldehyde), cryopreserved andprocessed for morphometric analysis of lesion extent.

Eleven normal mature baboons (Paio anubis) were studied. Six animalsreceived DEGR-FVIIa infusions (50 μg/kg/hr) and the remaining five werecontrol animals that did not receive DEGR-FVIIa. The animals weredewormed and observed to be disease-free for three months prior to use.All procedures were approved by the Institutional Animal Care and UseCommittee and were in compliance with procedures and methods outlined byNIH Guide for the Care and Use of Laboratory Animals, as well as theAnimal Welfare Act and related institutional policies. Invasiveprocedures were carried out under halothane anesthesia after inductionby ketamine (10 mg/kg intramuscularly) and valium (0.5 mg/kgintravenously). For subsequent short-term immobilization in performingexperimental procedures postoperatively, ketamine hydrochloride (5-20mg/kg intramuscularly) was used.

Carotid endarterectomy was performed through a midline neck incisionusing the technique of Hanson et al., Hypertension 18:1170-I-176 (1991)and Krupski et al., Circulation 84:1749-1757 (1991), incorporated hereinby reference. Endarterectomy was used as a vascular injury model becauseof its clinical relevance, and because VLF induced by endarterectomy ofnormal arteries has been shown to be reproducible. In brief, the commoncarotid artery was dissected free of surrounding tissues from theclavicle proximally to the carotid bifurcation distally. The commoncarotid artery was cross-clamped using atraumatic vascular clamps placedat each end of the exposed vessel three minutes after a bolus injectionof heparin sulfate (100 U/kg intravenously; Elkins-Simm Inc., CherryHill, N.J.) and divided 1 cm proximal to the distal crossclamp. Theproximal arterial segment was then everted over curved forceps. Aftermaximal eversion was obtained, a pair of polypropylene stay sutures(7-0) was placed on either side proximally and a second pair placeddistally in the lumen-exposed segment. The endarterectomy was thenperformed beginning 1 cm from the divided end of the everted vesselsegment and continued for a measured distance of 1 cm. This procedureinvolves mechanical removal of the normal intima and a partial thicknessof media using forceps and a surgical microscope (32×magnification).Following endarterectomy, the vessel was returned to its normalconfiguration, and an end-to-end anastomosis performed with 7-0polypropylene suture and continuous technique under 2.5-foldmagnification, and the wound closed in layers.

For morphometric analysis of VLF, sections embedded in paraffin andstained for connective tissue components (collagen, elastin) and withhematoxylin-eosin, were evaluated using a Zeiss Photoscope coupled withimage analysis system (Thomas Optical Measurement Systems, Columbus,Ga.) consisting of high resolution (580 lines) CCD microscope cameracoupled to a high resolution (700 lines) monitor, an IBM 386 chip, 80 MBcomputer with high resolution graphics digitablet for image acquisitionand storage. Quantitative image analysis was performed using amorphometric software driver (Optimas, Bioscan, Inc., Edmonds, Wash.).Arterial cross-sections were analyzed with respect to the total area ofneointimal proliferative lesion and corresponding area of arterialmedia. For statistical analysis, comparisons between groups were madeusing the Student's t test (two tailed) for paired and unpaired data.

The results showed that the intimal area was significantly decreased inthe animals treated with DEGR-Factor VIIa for seven days and studied at30 days as compared to control animals who had undergone the samevascular injury but who did not obtain any DEGR-Factor VIIa (FIG. 4). Asimilar result was found in the animal treated with DEGR-Factor VIIa for30 days and examined at 30 days.

Preliminary studies with a ballon angiographic brachial artery modelsuggested no measurable benefit of DEGR-Factor VIIa therapy. This model,however, has not been shown in baboons to be a prothrombotic model inwhich tissue factor plays a key role.

Studies with the femoral artery balloon injury in the baboon did show astatistically significant benefit from DEGR-Factor VIIa as compared tocontrols, as shown in FIG. 5.

It is evident from the foregoing that compositions of Factor VII or VIIahaving modified catalytic sites are provided which are able to bindtissue factor yet are substantially unable to activate Factors X and IX.As modified Factor VII acts specifically to interrupt the clottingcascade without degrading or consuming clotting factors, administrationof modified Factor VII preparations will be accompanied by fewerundesirable side effects than experienced with current therapies.Further, the modified Factor VII described herein may readily beproduced by recombinant means. Thus efficacy, convenience and economicsof lower dosages and less frequent administration, and a relative lackof toxicity are among the advantages conferred by the compositions ofthe present invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 4                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2422 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: N                                                    -     (iv) ANTI-SENSE: N                                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 28..1420                                              #/codon.sub.-- start= 28RMATION:                                                             /product=- # "Factor VII"                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - CCTCCCGACA ATACAGGGGC AGCACTGCAG AGATTTCATC ATG GTC TC - #C CAG GCC           55                                                                          #        Met Val Ser Gln Ala                                                  #-35                                                                          - CTC AGG CTC CTC TGC CTT CTG CTT GGG CTT CA - #G GGC TGC CTG GCT GCA          103                                                                          Leu Arg Leu Leu Cys Leu Leu Leu Gly Leu Gl - #n Gly Cys Leu Ala Ala           20                                                                            - GTC TTC GTA ACC CAG GAG GAA GCC CAC GGC GT - #C CTG CAC CGG CGC CGG          151                                                                          Val Phe Val Thr Gln Glu Glu Ala His Gly Va - #l Leu His Arg Arg Arg           - CGC GCC AAC GCG TTC CTG GAG GAG CTG CGG CC - #G GGC TCC CTG GAG AGG          199                                                                          Arg Ala Asn Ala Phe Leu Glu Glu Leu Arg Pr - #o Gly Ser Leu Glu Arg           #   15                                                                        - GAG TGC AAG GAG GAG CAG TGC TCC TTC GAG GA - #G GCC CGG GAG ATC TTC          247                                                                          Glu Cys Lys Glu Glu Gln Cys Ser Phe Glu Gl - #u Ala Arg Glu Ile Phe           #                 30                                                          - AAG GAC GCG GAG AGG ACG AAG CTG TTC TGG AT - #T TCT TAC AGT GAT GGG          295                                                                          Lys Asp Ala Glu Arg Thr Lys Leu Phe Trp Il - #e Ser Tyr Ser Asp Gly           #             45                                                              - GAC CAG TGT GCC TCA AGT CCA TGC CAG AAT GG - #G GGC TCC TGC AAG GAC          343                                                                          Asp Gln Cys Ala Ser Ser Pro Cys Gln Asn Gl - #y Gly Ser Cys Lys Asp           #         60                                                                  - CAG CTC CAG TCC TAT ATC TGC TTC TGC CTC CC - #T GCC TTC GAG GGC CGG          391                                                                          Gln Leu Gln Ser Tyr Ile Cys Phe Cys Leu Pr - #o Ala Phe Glu Gly Arg           #     75                                                                      - AAC TGT GAG ACG CAC AAG GAT GAC CAG CTG AT - #C TGT GTG AAC GAG AAC          439                                                                          Asn Cys Glu Thr His Lys Asp Asp Gln Leu Il - #e Cys Val Asn Glu Asn           # 95                                                                          - GGC GGC TGT GAG CAG TAC TGC AGT GAC CAC AC - #G GGC ACC AAG CGC TCC          487                                                                          Gly Gly Cys Glu Gln Tyr Cys Ser Asp His Th - #r Gly Thr Lys Arg Ser           #               110                                                           - TGT CGG TGC CAC GAG GGG TAC TCT CTG CTG GC - #A GAC GGG GTG TCC TGC          535                                                                          Cys Arg Cys His Glu Gly Tyr Ser Leu Leu Al - #a Asp Gly Val Ser Cys           #           125                                                               - ACA CCC ACA GTT GAA TAT CCA TGT GGA AAA AT - #A CCT ATT CTA GAA AAA          583                                                                          Thr Pro Thr Val Glu Tyr Pro Cys Gly Lys Il - #e Pro Ile Leu Glu Lys           #       140                                                                   - AGA AAT GCC AGC AAA CCC CAA GGC CGA ATT GT - #G GGG GGC AAG GTG TGC          631                                                                          Arg Asn Ala Ser Lys Pro Gln Gly Arg Ile Va - #l Gly Gly Lys Val Cys           #   155                                                                       - CCC AAA GGG GAG TGT CCA TGG CAG GTC CTG TT - #G TTG GTG AAT GGA GCT          679                                                                          Pro Lys Gly Glu Cys Pro Trp Gln Val Leu Le - #u Leu Val Asn Gly Ala           160                 1 - #65                 1 - #70                 1 -       #75                                                                           - CAG TTG TGT GGG GGG ACC CTG ATC AAC ACC AT - #C TGG GTG GTC TCC GCG          727                                                                          Gln Leu Cys Gly Gly Thr Leu Ile Asn Thr Il - #e Trp Val Val Ser Ala           #               190                                                           - GCC CAC TGT TTC GAC AAA ATC AAG AAC TGG AG - #G AAC CTG ATC GCG GTG          775                                                                          Ala His Cys Phe Asp Lys Ile Lys Asn Trp Ar - #g Asn Leu Ile Ala Val           #           205                                                               - CTG GGC GAG CAC GAC CTC AGC GAG CAC GAC GG - #G GAT GAG CAG AGC CGG          823                                                                          Leu Gly Glu His Asp Leu Ser Glu His Asp Gl - #y Asp Glu Gln Ser Arg           #       220                                                                   - CGG GTG GCG CAG GTC ATC ATC CCC AGC ACG TA - #C GTC CCG GGC ACC ACC          871                                                                          Arg Val Ala Gln Val Ile Ile Pro Ser Thr Ty - #r Val Pro Gly Thr Thr           #   235                                                                       - AAC CAC GAC ATC GCG CTG CTC CGC CTG CAC CA - #G CCC GTG GTC CTC ACT          919                                                                          Asn His Asp Ile Ala Leu Leu Arg Leu His Gl - #n Pro Val Val Leu Thr           240                 2 - #45                 2 - #50                 2 -       #55                                                                           - GAC CAT GTG GTG CCC CTC TGC CTG CCC GAA CG - #G ACG TTC TCT GAG AGG          967                                                                          Asp His Val Val Pro Leu Cys Leu Pro Glu Ar - #g Thr Phe Ser Glu Arg           #               270                                                           - ACG CTG GCC TTC GTG CGC TTC TCA TTG GTC AG - #C GGC TGG GGC CAG CTG         1015                                                                          Thr Leu Ala Phe Val Arg Phe Ser Leu Val Se - #r Gly Trp Gly Gln Leu           #           285                                                               - CTG GAC CGT GGC GCC ACG GCC CTG GAG CTC AT - #G GTC CTC AAC GTG CCC         1063                                                                          Leu Asp Arg Gly Ala Thr Ala Leu Glu Leu Me - #t Val Leu Asn Val Pro           #       300                                                                   - CGG CTG ATG ACC CAG GAC TGC CTG CAG CAG TC - #A CGG AAG GTG GGA GAC         1111                                                                          Arg Leu Met Thr Gln Asp Cys Leu Gln Gln Se - #r Arg Lys Val Gly Asp           #   315                                                                       - TCC CCA AAT ATC ACG GAG TAC ATG TTC TGT GC - #C GGC TAC TCG GAT GGC         1159                                                                          Ser Pro Asn Ile Thr Glu Tyr Met Phe Cys Al - #a Gly Tyr Ser Asp Gly           320                 3 - #25                 3 - #30                 3 -       #35                                                                           - AGC AAG GAC TCC TGC AAG GGG GAC AGT GGA GG - #C CCA CAT GCC ACC CAC         1207                                                                          Ser Lys Asp Ser Cys Lys Gly Asp Ser Gly Gl - #y Pro His Ala Thr His           #               350                                                           - TAC CGG GGC ACG TGG TAC CTG ACG GGC ATC GT - #C AGC TGG GGC CAG GGC         1255                                                                          Tyr Arg Gly Thr Trp Tyr Leu Thr Gly Ile Va - #l Ser Trp Gly Gln Gly           #           365                                                               - TGC GCA ACC GTG GGC CAC TTT GGG GTG TAC AC - #C AGG GTC TCC CAG TAC         1303                                                                          Cys Ala Thr Val Gly His Phe Gly Val Tyr Th - #r Arg Val Ser Gln Tyr           #       380                                                                   - ATC GAG TGG CTG CAA AAG CTC ATG CGC TCA GA - #G CCA CGC CCA GGA GTC         1351                                                                          Ile Glu Trp Leu Gln Lys Leu Met Arg Ser Gl - #u Pro Arg Pro Gly Val           #   395                                                                       #TGGCCTGTGG             1396C TAG C CCAGCAGCCC                                Leu Leu Arg Ala Pro Phe Pro                                                   400                 4 - #05                                                   - AGAGAAAGCC AAGGCTGCGT CGAACTGTCC TGGCACCAAA TCCCATATAT TC - #TTCTGCAG       1456                                                                          - TTAATGGGGT AGAGGAGGGC ATGGGAGGGA GGGAGAGGTG GGGAGGGAGA CA - #GAGACAGA       1516                                                                          - AACAGAGAGA GACAGAGACA GAGAGAGACT GAGGGAGAGA CTCTGAGGAC AT - #GGAGAGAG       1576                                                                          - ACTCAAAGAG ACTCCAAGAT TCAAAGAGAC TAATAGAGAC ACAGAGATGG AA - #TAGAAAAG       1636                                                                          - ATGAGAGGCA GAGGCAGACA GGCGCTGGAC AGAGGGGCAG GGGAGTGCCA AG - #GTTGTCCT       1696                                                                          - GGAGGCAGAC AGCCCAGCTG AGCCTCCTTA CCTCCCTTCA GCCAAGCCCC AC - #CTGCACGT       1756                                                                          - GATCTGCTGG CCCTCAGGCT GCTGCTCTGC CTTCATTGCT GGAGACAGTA GA - #GGCATGAA       1816                                                                          - CACACATGGA TGCACACACA CACACGCCAA TGCACACACA CAGAGATATG CA - #CACACACG       1876                                                                          - GATGCACACA CAGATGGTCA CACAGAGATA CGCAAACACA CCGATGCACA CG - #CACATAGA       1936                                                                          - GATATGCACA CACAGATGCA CACACAGATA TACACATGGA TGCACGCACA TG - #CCAATGCA       1996                                                                          - CGCACACATC AGTGCACACG GATGCACAGA GATATGCACA CACCGATGTG CG - #CACACACA       2056                                                                          - GATATGCACA CACATGGATG AGCACACACA CACCAAGTGC GCACACACAC CG - #ATGTACAC       2116                                                                          - ACACAGATGC ACACACAGAT GCACACACAC CGATGCTGAC TCCATGTGTG CT - #GTCCTCTG       2176                                                                          - AAGGCGGTTG TTTAGCTCTC ACTTTTCTGG TTCTTATCCA TTATCATCTT CA - #CTTCAGAC       2236                                                                          - AATTCAGAAG CATCACCATG CATGGTGGCG AATGCCCCCA AACTCTCCCC CA - #AATGTATT       2296                                                                          - TCTCCCTTCG CTGGGTGCCG GGCTGCACAG ACTATTCCCC ACCTGCTTCC CA - #GCTTCACA       2356                                                                          - ATAAACGGCT GCGTCTCCTC CGCACACCTG TGGTGCCTGC CACCCAAAAA AA - #AAAAAAAA       2416                                                                          #         2422                                                                - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 444 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Val Ser Gln Ala Leu Arg Leu Leu Trp Le - #u Leu Leu Gly Leu Gln         25                                                                            - Gly Cys Leu Ala Ala Val Phe Val Thr Gln Gl - #u Glu Ala His Gly Val         10                                                                            - Leu His Arg Arg Arg Arg Ala Asn Ala Phe Le - #u Glu Glu Leu Arg Pro         #   10                                                                        - Gly Ser Leu Glu Arg Glu Cys Lys Glu Glu Gl - #n Cys Ser Phe Glu Glu         #                 25                                                          - Ala Arg Glu Ile Phe Lys Asp Ala Glu Arg Th - #r Lys Leu Phe Trp Ile         #             40                                                              - Ser Tyr Ser Asp Gly Asp Gln Cys Ala Ser Se - #r Pro Cys Gln Asn Gly         #         55                                                                  - Gly Ser Cys Lys Asp Gln Leu Gln Ser Tyr Il - #e Cys Phe Cys Leu Pro         #     70                                                                      - Ala Phe Glu Gly Arg Asn Cys Glu Thr His Ly - #s Asp Asp Gln Leu Ile         # 90                                                                          - Cys Val Asn Glu Asn Gly Gly Cys Glu Gln Ty - #r Cys Ser Asp His Thr         #                105                                                          - Gly Thr Lys Arg Ser Cys Arg Cys His Glu Gl - #y Tyr Ser Leu Leu Ala         #           120                                                               - Asp Gly Val Ser Cys Thr Pro Thr Val Glu Ty - #r Pro Cys Gly Lys Ile         #       135                                                                   - Pro Ile Leu Glu Lys Arg Asn Ala Ser Lys Pr - #o Gln Gly Arg Ile Val         #   150                                                                       - Gly Gly Lys Val Cys Pro Lys Gly Glu Cys Pr - #o Trp Gln Val Leu Leu         155                 1 - #60                 1 - #65                 1 -       #70                                                                           - Leu Val Asn Gly Ala Gln Leu Cys Gly Gly Th - #r Leu Ile Asn Thr Ile         #               185                                                           - Trp Val Val Ser Ala Ala His Cys Phe Asp Ly - #s Ile Lys Asn Trp Arg         #           200                                                               - Asn Leu Ile Ala Val Leu Gly Glu His Asp Le - #u Ser Glu His Asp Gly         #       215                                                                   - Asp Glu Gln Ser Arg Arg Val Ala Gln Val Il - #e Ile Pro Ser Thr Tyr         #   230                                                                       - Val Pro Gly Thr Thr Asn His Asp Ile Ala Le - #u Leu Arg Leu His Gln         235                 2 - #40                 2 - #45                 2 -       #50                                                                           - Pro Val Val Leu Thr Asp His Val Val Pro Le - #u Cys Leu Pro Glu Arg         #               265                                                           - Thr Phe Ser Glu Arg Thr Leu Ala Phe Val Ar - #g Phe Ser Leu Val Ser         #           280                                                               - Gly Trp Gly Gln Leu Leu Asp Arg Gly Ala Th - #r Ala Leu Glu Leu Met         #       295                                                                   - Val Leu Asn Val Pro Arg Leu Met Thr Gln As - #p Cys Leu Gln Gln Ser         #   310                                                                       - Arg Lys Val Gly Asp Ser Pro Asn Ile Thr Gl - #u Tyr Met Phe Cys Ala         315                 3 - #20                 3 - #25                 3 -       #30                                                                           - Gly Tyr Ser Asp Gly Ser Lys Asp Ser Cys Ly - #s Gly Asp Ser Gly Gly         #               345                                                           - Pro His Ala Thr His Tyr Arg Gly Thr Trp Ty - #r Leu Thr Gly Ile Val         #           360                                                               - Ser Trp Gly Gln Gly Cys Ala Thr Val Gly Hi - #s Phe Gly Val Tyr Thr         #       375                                                                   - Arg Val Ser Gln Tyr Ile Glu Trp Leu Gln Ly - #s Leu Met Arg Ser Glu         #   390                                                                       - Pro Arg Pro Gly Val Leu Leu Arg Ala Pro Ph - #e Pro                         395                 4 - #00                 4 - #05                           - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: N                                                    -     (iv) ANTI-SENSE: N                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #21                CCCT T                                                     - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 15 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: N                                                    -     (iv) ANTI-SENSE: N                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #    15                                                                       __________________________________________________________________________

What is claimed is:
 1. A method for inhibiting vascular restenosis thatis associated with acute vascular injury in an individual, comprisingadministering to the individual a therapeutically effective amount of acomposition which comprises a pharmacologically acceptable Factor VIIhaving at least one covalent modification in its catalytic center, whichmodification inhibits the ability of the modified Factor VII to activateplasma Factor X or IX.
 2. The method of claim 1, wherein the Factor VIImodification is produced by reaction of the Factor VII with a serineprotease inhibitor.
 3. The method of claim 2, wherein the proteaseinhibitor is an organophosphor compound, a sulfanyl fluoride, a peptidehalomethyl ketone, or an azapeptide.
 4. The method of claim 3, whereinthe protease inhibitor is a peptide halomethyl ketone selected fromDansyl-Phe-Pro-Arg chloromethyl ketone or Dansyl-Glu-Gly-Argchloromethyl ketone.
 5. The method of claim 1, wherein the Factor VIImodification comprises at least one amino acid substitution or deletionin a catalytic triad of Ser, Asp, and His.
 6. The method of claim 1,wherein the restenosis is secondary to mechanical injury to an artery.7. The method of claim 6, wherein the mechanical injury is caused byballoon angioplasty, endarterectomy, reductive atherectomy, stentplacement, laser therapy or rotablation.
 8. The method of claim 6,wherein the composition is administered to the individual within 24hours prior to the mechanical injury to said artery.
 9. The method ofclaim 6, wherein the composition is further administered to theindividual subsequent to the mechanical injury to said artery.
 10. Themethod of claim 1, wherein the restenosis occurs in a vascular graft,stent, bypass graft or organ transplant.
 11. The method of claim 10,wherein the composition is administered to the individual within 24hours prior to placement of said graft, stent, bypass graft or organtransplant.
 12. The method of claim 10, wherein the composition isadministered to the individual subsequent to placement of said graft,stent, bypass graft or organ transplant.
 13. The method of claim 1,comprising further administering to said individual tissue plasminogenactivator or streptokinase.
 14. The method of claim 1, wherein theFactor VII is human Factor VII.
 15. A method for inhibiting vascularrestenosis in a patient comprising administering to the patient atherapeutically effective dose of a composition comprising human FactorVII covalently modified in its catalytic center by a protease inhibitorselected from an organophosphor compound, a sulfanyl fluoride, a peptidehalomethyl ketone, or an azapeptide, and said modified human Factor VIIinhibits the ability of the modified Factor VII to activate human plasmaFactor X or IX.
 16. The method of claim 15, wherein the proteaseinhibitor is a peptide halomethyl ketone.
 17. The method of claim 15,wherein the modified human Factor VII is administered at the site of thevascular restenosis.
 18. The method of claim 15, wherein the modifiedhuman Factor VII is administered systemically.
 19. The method of claim15, wherein the modified human Factor VII is administered in an amountfrom 1 mg to 200 mg.